Research Article | | Peer-Reviewed

Molecular Characterization of Gram Negative Bacteria Involved in Sepsis Among Under Five Children in Akwa Ibom State Nigeria

Received: 18 April 2025     Accepted: 28 April 2025     Published: 11 September 2025
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Abstract

Sepsis is a life threatening medical emergency, Gram negative bacteria the principal causes of sepsis are seen in higher proportions among pediatrics populations and are mostly antibiotic resistance organisms. This study was carried out to determine antibiotic resistance of Gram negative bacteria, antibiotic resistance genes involve in sepsis among under five children in Akwa Ibom State, Nigeria. A hospital-based descriptive observational study of neonates with or without clinical features of sepsis. The subjects were children seen in General Hospital Ikot Ekpene, University Teaching Hospital Uyo and Immanuel Hospital Eket. A total of 180 children were sampled (60 from each hospital). A two milliliters (2 ml) sterile syringe with a 23gauge needle was used to collect blood sample aseptically from the vein of the arm of the child, inoculated on thioglycollate broth and subculture on MaConkey, blood and chocolate agar. Gram staining, biochemical characterization, antimicrobial susceptibility and resistance of Gram negative bacteria, their resistance genes were done. Of the 180 children, 123 tested positive for bacterial infections. Escherichia coli 25(13.9%), Proteus mirabilis 19(10.6%), Pseudomonas aeruginosa 15(8.3%), Klebsiella pneumoniae 12(6.7%), Serratia ficaria 9(5.0%), Rhizobium radiobacter 8(4.4%), Klebsiella oxytoca 7(3.9%), Chromobacterium violaceum 7(3.9%), Serratia marcescens 5(2.8%), Escherichia fergusonii 4(2.2%), Pseudomonas luteola 3(1.7%), Burkholderia cepacia 3(1.7%), Achromobacter xylosoxidans 3(1.7%), Burkholderia vietnamiensis 2(1.1%) and Serratia odorifera 1(0.6%). Pseudomonas aeruginosa was resistance to the 12 antibiotic used 12(100%), Three isolates were finally selected for molecular analysis, E. coli, P. aeruginosa, K. pneumoniae acquire blaSHV, PAGS, PASS, Cnf1 and hlyC genes, blaTEM amplify Pseudomonas and Klebsiella, FimH amplify only Klebsiella. Gram negative bacteria develop antibiotic resistance which poses a significant challenge in treating infections caused by this organism emphasizing the importance of responsible antibiotic use to mitigate further development of resistance.

Published in International Journal of Microbiology and Biotechnology (Volume 10, Issue 3)
DOI 10.11648/j.ijmb.20251003.15
Page(s) 111-130
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2025. Published by Science Publishing Group

Keywords

Sepsis, Antibiotic, Resistance Genes, Gram Negative Bacteria, Children

1. Introduction
Background of the Study
Sepsis is an overwhelming response to an infection and leads to tissue damage, organ failure and even death (Basco, 2021) . Septicemia is an infection that occurs when bacteria circulate and actively multiply in the bloodstream and can also be refer to as blood poisoning (Basco, 2021; CDC, 2023) . Globally, sepsis is one of the major causes of morbidity and mortality in children, almost half of all global sepsis cases occurred among children, with an estimated 20 million cases and 2.9 million global deaths in children under five years of age (Rudd et al., 2020) . Annually over 30 million people are affected worldwide, 6 million deaths, 3 million newborn and 1.2 million children and about 3-10 deaths are due to neonatal sepsis caused by resistant pathogens (Bonet et al., 2018) . The rising prevalence of multidrug-resistant (MDR) Gram negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Olowo-Okere et al., (2020); Tessema et al., (2021); Breijyeh et al., (2020), in Tanzania, Carroll et al., (2016); Ethiopia, Sisay et al., (2019) also report Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis has significantly contributed to increased mortality rates in health care facilities. In Nigeria, studies indicate that the prevalence rate of sepsis is 3-10 times higher in preterm than in full-term neonates and also higher in low birth weight than normal weight babies (Olorukooba et al., 2020) . Medugu et al., (2018) observe increase sepsis in newborn due to poor parental education and low income factors. Onubogu and West, (2022) observe that male has predominant number of sepsis compare to female. Sepsis can be the clinical manifestation of infections acquired both in health care facilities [caused by surgical site infection (SSI), ventilator-associated pneumonia (VAP), catheter-associated urinary tract infections (CAUTI), central line-associated bloodstream infection (CLABSI), methicillin-resistant Staphylococcus aureus (MRSA)] and community settings. Gram negative bacteria is the most frequently infections of the respiratory, gastrointestinal, genitourinary, liver and biliary tracts that enter the circulation and is mostly seen among children under 5 years and elderly (older than 65 years) which is associated with an increased morbidity and death (WHO, 2022) . Example of Gram negative bacteria seen in higher proportions among pediatrics populations that are mostly antibiotic resistance organisms are; Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (Ogbolu et al., 2019; WHO, 2022) . Others are Enterobacter aerogenes, Acinetobacter baumanii, Serratia spp., Neisseria spp., Burkholderia spp., Achromobacter spp., Helicobacter pylori Owowo et al., (2019) , and Chlamydia trachomatis) (WHO, 2022) . Those at risk of sepsis are; Neonates, elderly, immunocompromised, pregnant women, hospitalized patients, recent surgery/procedure, indwelling catheter, diabetis mellitus, haemodialysis, presence of malignancy/anticancer medication and drugs/alcohol use etc. (Lawn et al., 2017) . Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species through mobile genetic elements, ultimately limiting treatments in low and middle-income countries (LMICs), are poorly characterized (Folgori et al., 2017) . Gram negative bacteria: the principal cause of sepsis is because Gram negative bacteria have much stronger membrane around the cell than Gram positive bacteria, making them much more difficult to treat (Ogbolu et al., 2019) . Also mechanism of resistance are mostly seen in Gram negative bacteria mediated by a mobile plasmid; β-lactamases in periplasmic space, over-expression of trans membrane efflux pump Lister et al., (2009) , loss of porins, antibiotic modifying enzymes, target site mutations, ribosomal mutation and mutation in lipopolysaccharide (Devi et al., 2018; CDC, 2019) . Due to increase in children’s death in South-Southern region of Nigeria and the financial implication incurred by the parent to serve the life of children, even with the increase in drug resistance motivated this study. The aim of the study is to characterize and determine the antibiotic resistance of Gram negative bacteria and antibiotic resistance genes of bacteria isolates involves in sepsis among under five children in Akwa Ibom State.
2. Materials and Methods
2.1. Study Area and Population
This 18 month study (June 2023-December 2024) was conducted in three senatorial districts of Akwa Ibom State, Nigeria: Ikot Ekpene (North West), Eket (South) and Uyo (North East). Three hospitals General Hospital Ikot Ekpene, Immanuel Hospital Eket and Teaching Hospital Uyo were selected. The study assessed all admitted children (0-5 years) with or without clinical signs of sepsis. Akwa Ibom, located in Nigeria’s south-south geopolitical zone, shares borders with Cross River, Rivers, and Abia States and the Atlantic Ocean. It has 31 local government areas, with a total land mass of 7,081km2 (7,739 sq. mil). Latitude 05° 00N of the equator and Longitude 07° 50E of the Greenish Meridian. Health infrastructure; hospitals, clinics, with Ibibio as the dominant language. The economy relies on trading, fishing, and palm oil exports. Study sites were selected for convenience and proximity to the laboratory for specimen analysis. The study population consisted of children (both male and female), children under five years of age (0-5) seen in general out-patient, gynaecological out-patient clinics, pediatric clinics and immunization unit in the hospital of study.
Figure 1. Map showing the three Senatorial Districts in Akwa Ibom State (NPC, 2011) .
2.2. Sample Collection and Processing
A total of 180 blood samples were aseptically collected using sterile syringes and transferred to the clinical microbiology laboratory within 24 hours for culture. Gram negative bacteria were isolated using thioglycollate broth, incubated at 35-37°C for 7 days, and subcultured on selective media (MacConkey, Blood and Chocolate agar).
2.3. Identification and Characterization
Bacterial isolates were identified using Gram staining and the Biomerieux VITEK 2 system. Biochemical characterization followed standard protocols to confirm bacterial species.
2.4. Antimicrobial Susceptibility Testing
The Kirby-Bauer disc diffusion method was used to assess antibiotic susceptibility according to CLSI guidelines (CLSI, 2020) . Antimicrobial susceptibility test was carried out using the Kirby-Bauer disc diffusion method. A colony of each isolate was grown on Muller Hinton broth. Broth culture was diluted with normal saline to obtain similar turbidity as 0.05 McFarland standards. A standardized microbial culture was spread on Muller Hinton agar to generate a lawn culture. All confirmed isolates was tested against twelve antibiotic agents a multi disks namely; [tetracycline (TET) 10µg, cotrimoxazole (COT) 25µg, gentamicin (GEN) 10µg, cefuroxime (CRX) 30µg, chloramphenicol (CHL) 10µg, ceftriaxone (CTR) 30µg, cefotaxime (CTX) 30µg, ciprofloxacin (CIP) 5µg, amikacin (AMK) 30µg, vancomycin (VAN) 30µg, ceftazidime (CPZ) 30µg, meropenem (MEM) 10µg]. A maximum of six antibiotic discs was aseptically placed on each agar plates containing isolated organism. 24-30mm apart, the center to center between disks on the same plates. All plates were left to stand for 1 hour on the bench at room temperature for pre-diffusion of antibiotics before incubation at 37°C for 24 hours. After incubation, the zones of inhibition was measured in millimeter (mm) with ruler, recorded and interpreted as susceptible, intermediate and resistant. Multidrug resistance (MDR) was determined using the multiple antibiotic resistances (MAR) index.
2.5. Molecular Analysis
The molecular analysis was carried out at Genomics Training Center and Laboratory Limited, Uyo, Akwa Ibom.
2.5.1. DNA Extraction
DNA extraction was performed using the Zymo Research Quick-DNA Fungal and Bacteria Kit). Bacteria culture was suspended in 200µl of ultrapure water; 750µl of BashingBead buffer was also added and transferred to a ZR BashingBead lysis tube. Lysis tubes were vortexed for about 5 minutes to dissociate the bacteria cells. Centrifuge the ZR Bashing Bead lysis tube in a microcentrifuge at 10,000g for 1 minute. Then transfer up to 400µl of the supernatant to a zymo-spin filter in a collection tube and centrifuge at 8,000g for 1 minute. Add 1,200µl of genomic lysis buffer to the filtrate in the collection tube from the last step. Transfer 800µl of the mixture from step 5 to a zymo-spin IICR column in a collection tube and centrifuge at 10,000g for 1 minute. Discard the flow through and repeat the last step. Then add 200µl DNA pre-wash buffer to the zymo-spin IICR column and centrifuge at 10,000g in a new collection tube for 1 minute. Add 500µl g-DNA wash buffers to the zymo-spin IICR column and centrifuge at 10,000g for 1 minute. Now transfer the zymo-spin IICR column to a clean 1.5 ml microcentrifuge tube and add 100µl DNA elution buffer directly to the column matrix, centrifuge at 10,000g for 30 seconds to elute the DNA.
2.5.2. DNA Quantification
DNA concentration was determined using a Spectrophotometer (Gene Quant Pro). The absorbance of total genomic DNA (gDNA) was quantified by measuring optical density (OD) at 260 nm and 280 nm.
Table 1. DNA Concentration and Purity of Bacteria Samples.

Sample ID

DNA Concentration (ug/ul)

DNA Purity (260/280)

A

311

1.82

B

395

1.89

C

361

1.80

2.5.3. Gel Electrophoresis
The presence and quality of gDNA was also evaluated by agarose gel electrophoresis. DNA was accessed on 1.5% agarose gel. Electrophoresis was conducted in a 1X TAE (Tris-base glacial acetic acid, EDTA) buffer at 120 volts for 20 minutes. The gel was stained with 3ul of ethidium bromide stain. The gel was visualized under an EV transilluminator (Accuris SmartDoc 2.0).
2.5.4. Polymerase Chain Reaction (PCR) Amplification of Genes
The PCR master mix contained PCR amplification buffer, MgCl, DMSO, DNTPs and Taq polymerase. Other reagents include 16S primers (forward and reverse), ultra-pure molecular biology grade water and template DNA. The PCR final reaction volume was made up to 20µl, using 4µl master mix (Solisbiodyne), 0.5µl forward and reverse primer, 1µl of sample DNA and 14µl nuclease free water.
Table 2. The Primer Sequences used for Amplification of Bla Genes.

Target

Primer

Primer Sequence (5̗’- 3’)

Bases

Product Size (bp)

Ref.

blaTEM

TEM-F

TCCGCTCATGAGACAATAACC

21

890

(Sturenburg et al., 2004)

.

TEM-R

TTGGTCTGACAGTTACCAATGC

22

890

(Sturenburg et al., 2004)

.

bla SHV

SHV-F

TGGTTATGCGTTATATTCGCC

21

868

(Pai et al., 1999)

.

SHV-R

GGTTAGCGTTGCCAGTGCT

19

868

(Pai et al., 1999)

.

blaCTX-M

CTX-F

TCTTCCAGAATAAGGAATCCC

21

909

(Sturenburg et al., 2004)

.

CTX-R

CCGTTTCCGCTATTACAAAC

20

909

(Sturenburg et al., 2004)

.

TEM (Temoniera), SHV (Sulf-hydryl), CTX-M (Cefotaximase-munich).
Table 3. Primer Sequences used for Detection of Carbapenemase Encoding Genes in Klebsiella pneumoniae.

Gene Type

Primer

Primer Sequence

Bases Product Size (bp)

Ref.

CARB KPC

CARB-F

5’ATTCGCTAAACTCGAACAG-3’19

1069

(Mlynarcik et al., 2016)

.

CARB-R

5’AAGAAAGCCCTTGAATGAG-3’19

1069

(Mlynarcik et al., 2016)

.

VIM-1

VIM-I-F

5’GAGCTCTTCTATCCTGGTG-3’19

1069

(Mlynarcik et al., 2016)

.

VIM-1-R

5’CTTGACAACTCATGAACGG-3’19

1069

(Mlynarciket al., 2016)

.

Table 4. The Primer Sequences used for Amplification of Pseudomonas spp. and Pseudomonas aeruginosa.

Primer

Primer Sequence (5̗’- 3’)

Bases

Product Size (bp)

Ref.

PAGS-F

GGGGGATCTTCGGACCTCA

19

910

(Spilker et al., 2004)

.

PAGS-R

TCCTTAGAGTGCCCACCCG

19

910

(Spilker et al., 2004)

.

PASS-F

GGGGGATCTTCGGACCTCA

19

930

(Spilker et al., 2004)

.

PASS-R

TCCTTAGAGTGCCCACCCG

19

910

(Spilker et al., 2004)

.

Table 5. Primer Sequences used for the Detection of Virulence Factors in Neonatal Beta-lactamase Encoding Gene.

Virulence Gene

Primer Sequence (5’-3’)

Bases Product size (bp)

Ref.

fimH

F: TGCAGAACGGATAAGCCGTGG

21 470

(Johnson and Stell, 2000)

.

R: GCAGTCACCTGCCCTCCGGTA

21 460

(Johnson and Stell, 2000)

.

cnfI

F: AAGATGGAGTTTCCTATGCAGGAG

24 498

(Johnson and Stell, 2000)

.

R: CATTCAGAGTCCTGCCCTCATTATT

25 498

(Johnson and Stell, 2000)

.

hlyC

F: AGGTTCTTGGGCATGTATCCT

21 556

(Bingen-Bidois et al., 2002)

.

R: TTGCTTTGCAGACTGCAGTGT

21 556

(Bingen-Bidois et al., 2002)

.

Fim H (type I fimbrae), cnf I (cytotoxic necrotizing factor I) and hly C (haemolysin)
2.5.5. PCR Conditions
PCR reaction conditions conducted at BIO-RAD thermocycler were the following: initial denaturation at 95°C for 30 seconds, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, initial elongation at 72°C for 1 minutes and final elongation at 72°C for 5 minutes. Amplicons were separated on 1.5% agarose gel electrophoresis for 20 minutes at 120 V. DNA ladder of 50 bp was used as molecular weight standard.
2.6. Quality Controls
Control experiments (positive and negative) were set-up to monitor the efficiency of the media, reagents and different biochemical test performed.
2.7. Ethical Approval and Consent to Participate
Ethics committee of Akwa Ibom State ministry of health with committee reference number AKHREC/8/05/23/152 provided ethical clearance for the study. The research was conducted in accordance with the declaration of Helsinki 1964. Participants’ privacy and confidentiality were assured; all data and results were handled and treated confidentially.
2.8. Data Analysis
All data analyses were performed using Statistical Package for Social Sciences (SPSS) Computer Software Version 22. Descriptive analyses using percentages and frequencies were used for presence of antibiotic resistance pattern of Gram negative isolates and resistance genes.
3. Results
A total of 180 samples from the three senatorial districts of Akwa Ibom State, 123 were positive and 57 negative. Ikot Ekpene, Uyo and Eket (39, 43, 41) respectively. The highest prevalence was seen in Teaching Hospital Uyo, followed by Immanuel Hospital Eket and the least was in General Hospital Ikot Ekpene. Escherichia coli had the highest percentage rate 25 (13.9%), Proteus mirabilis 19 (10.6%), Pseudomonas aeruginosa 15 (8.3%), Klebsiella pneumoniae 12 (6.7%), Serratia ficaria 9 (5.0%), Rhizobium radiobacter 8 (4.4%), K. oxytoca 7 (3.9%), Chromobacterium violaceum 7 (3.9%), Serratia marcescens 5 (2.8%), Escherichia fergusonii 4 (2.2%), Pseudomonas luteola 3 (1.7%), Burkholderia cepacia 3 (1.7%), Achromobacter xylosoxidans 3 (1.7%), Burkholderia vietnamiensis 2 (1.1%) and the least was seen in S. odorifera 1 (0.6%) (Table 6).
Table 6. Determination of Gram Negative Bacteria Isolated from under five Children in General, Teaching and Immanuel Hospital (Ikot Ekpene, Uyo and Eket).

Gram negative bacteria

Nos. of tested bacteria (n), (n/N)% in (General Hospital)

Nos. of tested bacteria (n), (n/N)% in (Teaching Hospital)

Nos. of tested bacteria (n), (n/N)% in (Immanuel Hospital)

Total nos. of tested bacteria (n), (n/N)%

Escherichia coli

8 (13.3)

3 (5.0)

14 (23.3)

25 (13.9)

Proteus mirabilis

5 (8.3)

8 (13.3)

6 (10.0)

19 (10.6)

Pseudomonas aeruginosa

6 (10.0)

9 (15.0)

0 (0)

15 (8.3)

K. pneumoniae

4 (6.7)

8 (13.3)

0 (0)

12 (6.7)

S. ficaria

3 (5.0)

3 (5.0)

3 (5.0)

9 (5.0)

Rhizobium radiobacter

4 (6.7)

1 (1.7)

3 (5.0)

8 (4.4)

Klebsiella oxytoca

1 (1.7)

2 (3.3)

4 (6.7)

7 (3.9)

Chromobacterium violaceum

0 (0)

5 (8.3)

2 (3.3)

7 (3.9)

Serratia marcescens

2 (3.3)

2 (3.3)

1 (1.7)

5 (2.8)

Escherichia fergusonii

1 (1.7)

0 (0)

3 (5.0)

4 (2.2)

P. luteola

1 (1.7)

0 (0)

2 (3.3)

3 (1.7)

Burkholderia cepacia

1 (1.7)

0 (0)

2 (3.3)

3 (1.7)

Achromobacter xylosoxidans

1 (1.7)

1 (1.7)

1 (1.7)

3 (1.7)

B. vietnamiensis

1 (1.7)

1 (1.7)

0 (0)

2 (1.1)

S. odorifera

1 (1.7)

0 (0)

0 (0)

1 (0.6)

Total

39 (65.0)

43 (71.7)

41 (68.3)

123 (68.3)

Percentage antibiotic susceptibility of Gram negative bacteria (E. coli, P. mirabilis, P. aeruginosa, K. pneumonia, S. ficaria) isolated from under five children (Table 7). The highest proportion of susceptibility was seen in tetracycline (TET), gentamicin (GEN), chloramphenicol (CHL), ciprofloxacin (CIP), amikacin (AMK) and vancomycin (VAN). The highest proportion of resistance was seen in tetracycline (TET), cotrimoxazole (COT), cefotaxime (CTX), cefuroxime (CRX), ceftriaxone (CTR), ceftazidime (CPZ), ciprofloxacin (CIP) and meropenem (MEM). Escherichia coli and Proteus mirabilis were 100% susceptible to amikacin, E. coli 100% resistance to cefotaxime and ceftazidime, P. mirabilis 100% resistance to ceftazidime. Pseudomonas aeruginosa was 40% susceptible to Vancomycin and 100% resistance to cefotaxime, cefuroxime, ceftriaxone, ceftazidime, meropenem. Klebsiella pneumoniae 100% susceptible to amikacin and 100% resistance to cefotaxime, ceftazidime, meropenem. Serratia ficaria was 100% susceptible to amikacin, tetracycline, vancomycin and resistance 100% to cefotaxime and meropenem.
Table 7. Percentage Antibiotic Susceptibility of Gram Negative Bacteria (E. coli, P. mirabilis, P. aeruginosa, K. pneumonia, S. ficaria) Isolated from under five Children in General, Teaching and Immanuel Hospital, (Ikot Ekpene, Uyo and Eket).

Nos. of isolated Gram negative bacteria

AP

TET (%)

COT (%)

GEN (%)

CRX (%)

CHL (%)

CTR (%)

CTX (%)

CIP (%)

AMK (%)

VAN (%)

CPZ (%)

MEM (%)

E. coli (25)

S

13 (52.0)

10 (40.0)

22 (88.0)

2 (8.0)

21 (84.0)

2 (8.0)

0

11 (44.0)

25 (100.0)

11 (44.0)

0

0

R

10 (40.0)

13 (52.0)

3 (12.0)

19 (76.0)

0

13 (52.0)

25 (100.0)

10 (40.0)

0

12 (48.0)

25 (100.0)

24 (96.0)

P. mirabilis (19)

S

12 (63.2)

11 (57.9)

16 (84.2)

1 (5.3)

5 (26.3)

3 (15.8)

0

7 (36.8)

19 (100.0)

14 (73.7)

0

1 (5.3)

R

5 (26.3)

8 (42.1)

2 (10.5)

15 (78.9)

6 (31.6)

13 (68.4)

18 (94.7)

4 (21.1)

0

3 (15.8)

19 (100.0)

18 (94.7)

P. aeruginosa (15)

S

1 (6.7)

0

5 (33.3)

0

2 (13.3)

0

0

5 (33.3)

5 (33.3)

6 (40.0)

0

0

R

13 (86.7)

13 (86.7)

10 (66.7)

15 (100.0)

13 (86.7)

15 (100.0)

15 (100.0)

10 (66.7)

10 (66.7)

9 (60.0)

15 (100.0)

15 (100.0)

K. pneumonia (12)

S

2 (16.7)

4 (33.3)

10 (83.3)

0

11 (91.7)

0

0

1 (8.3)

12 (100.0)

8 (66.7)

0

0

R

10 (83.3)

8 (66.7)

1 (8.3)

11 (91.7)

0

11 (91.7)

12 (100.0)

11 (91.7)

0

4 (33.3)

12 (100.0)

12 (100.0)

S. ficaria (9)

S

9 (100.0)

8 (88.9)

8 (88.9)

4 (44.4)

6 (66.7)

0

0

8 (88.9)

9 (100.0)

9 (100.0)

1 (11.1)

0

R

0

0

1 (11.1)

3 (33.3)

2 (22.2)

8 (88.9)

9 (100.0)

1 (11.1)

0

0

7 (77.8)

9 (100.0)

Key: Tetracycline (TET) 10µg, Cotrimoxazole (COT) 25µg, Gentamicin (GEN) 10µg, Amikacin (AMK) 30µg, Vancomycin (VAN) 30µg, Cefuroxime (CRX) 30µg, Ceftriaxone (CTR) 30µg, Cefotaxime (CTX) 30µg, Ceftazidime (CPZ) 30µg, Chloramphenicol (CHL) 10µg, Ciprofloxacin (CIP) 5µg, Meropenem (MEM) 10µg. AP - Antibiotic pattern, S - Susceptibility, R – resistance.
Percentage antibiotic susceptibility of Gram negative bacteria (R. radiobacter, K. oxytoca, C. violaceum, S. marcescens, E. fergusonii) (Table 8). Rhizobium radiobacter was 100% susceptible to AMK, CIP, GEN and resistance 100% to CTX, CTR. Klebsiella oxytoca susceptible 100% to CHL and resistance to TET, CTX, CTR, CPZ, CIP, MEM. Chromobacterium violaceum 100% susceptible to TET, CIP, AMK and resistance to CTX, CTR, CPZ, MEM. Serratia marcescens 100% susceptible to AMK, CHL, GEN and resistance to CTX, MEM. Escherichia fergusonii susceptible 100% to AMK, TET, GEN and resistance to CTX, CPZ.
Table 8. Percentage Antibiotic Susceptibility of Gram Negative Bacteria (R. radiobacter, K. oxytoca, C. violaceum, S. marcescens, E. fergusonii) Isolated from under five Children in General, Teaching and Immanuel Hospital, (Ikot Ekpene, Uyo and Eket).

Nos. of isolated Gram negative bacteria

AP

TET (%)

COT (%)

GEN (%)

CRX (%)

CHL (%)

CTR (%)

CTX (%)

CIP (%)

AMK (%)

VAN (%)

CPZ (%)

MEM (%)

R. radiobacter (8)

S

3 (37.5)

4 (50.0)

8 (100.0)

0

7 (87.5)

0

0

8 (100.0)

8 (100.0)

3 (37.5)

1 (11.1)

0

R

3 (37.5)

3 (37.5)

0

5 (62.5)

1 (12.5)

8 (100.0)

8 (100.0)

0

0

0

7 (77.8)

9 (100.0)

K. oxytoca (7)

S

0

1 (14.3)

1 (14.3)

0

7 (100.0)

0

0

0

1 (14.3)

6 (85.7)

0

0

R

7 (100.0)

6 (85.7)

6 (85.7)

6 (85.7)

0

7 (100.0)

7 (100.0)

7 (100.0)

3 (42.9)

1 (14.3)

7 (100.0)

7 (100.0)

C. violaceum (7)

S

7 (100.0)

3 (42.9)

6 (85.7)

0

4 (57.1)

0

0

7 (100.0)

7 (100.0)

2 (28.6)

0

0

R

0

3 (42.9)

1 (14.3)

6 (85.7)

1 (14.3)

7 (100.0)

7 (100.0)

0

0

1 (14.3)

7 (100.0)

7 (100.0)

S. marcescens (5)

S

4 (80.0)

2 (40.0)

5 (100.0)

0

5 (100.0)

0

0

4 (80.0)

5 (100.0)

4 (80.0)

0

0

R

0

3 (60.0)

0

4 (80.0)

0

4 (80.0)

5 (100.0)

0

0

1 (20.0)

4 (80.0)

5 (100.0)

E. fergusonii (4)

S

4 (100.0)

1 (25.0)

4 (100.0)

0

3 (75.0)

0

0

3 (75.0)

4 (100.0)

3 (75.0)

0

0

R

0

1 (25.0)

0

3 (75.0)

0

3 (75.0)

4 (1000)

0

0

1 (25.0)

4 (100.0)

3 (75.0)

Key: Tetracycline (TET) 10µg, Cotrimoxazole (COT) 25µg, Gentamicin (GEN) 10µg, Amikacin (AMK) 30µg, Vancomycin (VAN) 30µg, Cefuroxime (CRX) 30µg, Ceftriaxone (CTR) 30µg, Cefotaxime (CTX) 30µg, Ceftazidime (CPZ) 30µg, Chloramphenicol (CHL) 10µg, Ciprofloxacin (CIP) 5µg, Meropenem (MEM) 10µg. AP - Antibiotic pattern, S - Susceptibility, R – resistance.
Percentage antibiotic susceptibility of Gram negative bacteria (P. luteola, B. cepacia, A. xylosoxidans, B. vietnamiensis, S. odorifera) isolated from under five children (Table 9). Pseudomonas luteola susceptible 100% to TET, GEN, CHL, AMK, VAN and resistance to CTX, CRX, CTR, MEM. Burkholderia cepacia susceptible 100% to CHL, CIP, AMK and resistance to CTX, CRX, CPZ, MEM. Achromobacter xylosoxidans susceptible 100% to CIP, CHL, AMK and resistance to TET, CTX, CRX, CTR, CPZ, MEM. Burkholderia vietnamiensis was 100% susceptible to AMK and resistance 100% to TET, COT, CTX, CRX, CTR, CPZ, MEM. Serratia odorifera susceptible 100% to CHL, TET, CIP, AMK, VAN and resistance 100% to COT, CTX, CRX, CTR, CPZ and MEM.
The determination of antibiotic resistance pattern of Gram negative isolates (E. coli, P. mirabilis, P. aeruginosa) obtained from sepsis in under five children (Table 10). Out of 25 isolates of E. coli, 2 were resistance to 9 (75%) cefuroxime (CRX), ceftriaxone (CTR), cefotaxime (CTX), tetracycline (TET), ciprofloxacin (CIP), cotrimoxazole (COT), vancomycin (VAN), gentamicin (GEN), meropenem (MEM), 8 (70%) CRX, CTX, ceftazidime (CPZ), TET, COT, CIP, amikacin (AMK), MEM and 4 (30%) CTR, CTX, CPZ, MEM antibiotics, 6 were resistance to 7 (60%) CRX, CTR, CTX, CPZ, COT, CIP, MEM, 5 were resistance to 6 (50%) CRX, CTX, CPZ, TET, VAN, MEM and 8 were resistance to 5 (40%) CRX, CTR, CTX, CPZ, MEM. All isolates were resistance to cephalosporins and carbapenems class of antibiotics.
P. mirabilis had 19 isolates, 2 were resistance to 8 (70%) CRX, CTR, CTX, CPZ, CIP, COT, chloramphenicol (CHL), MEM, 11 were resistance to 6 (50%) CTX, CPZ, TET, COT, VAN, MEM, 5 to 5 (40%) CRX, CTX, CPZ, CHL, MEM, 1 to 4 (30%) CRX, CTX, CPZ, MEM. All isolates were resistance to cephalosporins and carbapenems.
P. aeruginosa had 15 isolates, 9 were resistance to 12 (100%) antibiotics CRX, CTR, CTX, CPZ, CIP, TET, COT, CHL, VAN, GEN, AMK, MEM, 1 to 11 (90%) CRX, CTR, CTX, CPZ, CIP, TET, COT, CHL, GEN, AMK, MEM, 7 (60%) CRX, CTR, CTX, CPZ, TET, COT, MEM, 6 (50%) CRX, CTR, CTX, CPZ, CHL, MEM and 5 (40%) CRX, CTR, CTX, CPZ, MEM, 2 to 8 (70%) CRX, CTR, CTX, CPZ, TET, COT, CHL, MEM and all the isolates were resistance to cephalosporins and carbapenems.
The determination of antibiotic resistance pattern of Gram negative isolates (K. pneumoniae, S. ficaria, R. radiobacter) obtained from sepsis in under five children (Table 11). K. pneumoniae had 12 isolates, 1 were resistance to 10 (80%) antibiotics CRX, CTR, CTX, CPZ, CIP, COT, TET, VAN, AMK, MEM, 8 (70%) CRX, CTR, CTX, CPZ, TET, COT, CIP, MEM, 6 (50%) CRX, CTR, CTX, CPZ, CIP, MEM and 5 (40%) CRX, CTR, CTX, CPZ, MEM, 4 to 9 (75%) CRX, CTR, CTX, CPZ, TET, COT, CIP, VAN, MEM and 7 (60%) CTR, CTX, CPZ, TET, COT, CIP, MEM. All the isolates were resistance to cephalosporins and carbapenems.
Out of the 9 isolates of S. ficaria, 2 were resistance to 6 (50%) antibiotics CTR, CTX, CPZ, CIP, GEN, MEM and 5 (40%) CTR, CTX, CPZ, CHL, MEM, 4 to 4 (30%) CTR, CTX, CPZ, MEM, 1 to 2 (20%) CTX, MEM. All the isolates were resistance to cephalosporins and carbapenems.
R. radiobacter had 8 isolates, 2 were resistance to 7 (60%) CRX, CTR, CTX, CPZ, TET, COT, MEM and 3 (30%) CTR, CTX, CHL, 3 to 6 (50%) CRX, CTR, CTX, CPZ, COT, MEM, 1 to 5 (40%) CRX, CTR, CTX, CPZ, MEM. All the isolates were resistance to cephalosporins.
The determination of antibiotic resistance pattern of Gram negative isolates (K. oxytoca, C. violaceum, S. marcescens, E. fergusonii, P. luteola) obtained from sepsis in under five children (Table 12). Out of 7 isolates of K. oxytoca 2 were resistance to 10 (80%) CRX, CTR, CTX, CPZ, TET, CIP, COT, VAN, AMK, MEM, 4 to 9 (75%) CRX, CTR, CTX, CPZ, TET, COT, CIP, GEN, MEM and 1 to 8 (70%) CTR, CTX, CPZ, TET, COT, CIP, GEN, MEM antibiotics. All isolates were resistance to cephalosporins, tetracyclines, sulfonamides, fluoroquinolones aminoglycosides and carbapenems.
Out of 7 isolates of C. violaceum 2 were resistance to 7 (60%) CRX, CTR, CTX, CPZ, VAN, GEN, MEM, 6 (50%) CRX, CTR, CTX, CPZ, COT, MEM and 5 (40%) CRX, CTR, CTX, CPZ, MEM, 1 to 4 (30%) CTR, CTX, CPZ, MEM. All isolates were resistance to cephalosporins and carbapenems.
S. marcescens had 5 isolates, 1 were resistance to 6 (50%) CRX, CTR, CTX, CPZ, COT, MEM and 4 (30%) CRX, CTR, CTX, MEM, 3 to 5 (40%) CTR, CTX, CPZ, COT, MEM. All isolates were resistance to cephalosporins and carbapenems.
E. fergusonii had 4 isolates, 1 were resistance to 6 (50%) CRX, CTR, CTX, CPZ, VAN, MEM and 5 (40%) CRX, CTR, CTX, CPZ, MEM, 2 to 4 (30%) CTR, CTX, CPZ, MEM. All isolates were resistance to cephalosporins and carbapenems.
Out of the 3 isolates of P. luteola, 1 were resistance to 6 (50%) CRX, CTR, CTX, CPZ, CIP, MEM and 2 to 5 (40%) CRX, CTR, CTX, CPZ, MEM. All isolates were resistance to cephalosporins and carbapenems.
The determination of antibiotic resistance pattern of Gram negative isolates (B. cepacia, A. xylosoxidans, B. vietnamiensis, S. odorifera) obtained from sepsis in under five children (Table 13). Out of the 3 isolates of B. cepacia, 2 were resistance to 7 (60%) CRX, CTX, CPZ, TET, COT, VAN, MEM and 1 to 5 (40%) CRX, CTR, CTX, CPZ, MEM. All isolates were resistance to cephalosporins and carbapenems.
Out of the 3 isolates of A. xylosoxidans, 1 were resistance to 9 (75%) CRX, CTR, CTX, CPZ, TET, COT, VAN, GEN, MEM, 8 (70%) CRX, CTR, CTX, CPZ, TET, GEN, VAN, MEM and 6 (50%) CRX, CTR, CTX, CPZ, TET, MEM. All isolates were resistance to cephalosporins, tetracyclines and carbapenems.
Out of the 2 isolates of B. vietnamiensis, 1 were resistance to 8 (70%) CRX, CTR, CTX, CPZ, TET, COT, VAN, MEM and 7 (60%) CRX, CTR, CTX, CPZ, TET, COT, MEM. All isolates were resistance to cephalosporins, tetracyclines, sulfonamides and carbapenems.
S. odorifera had 1 isolates which was resistance to 6 (50%) CTR, CTX, CRX, CPZ, COT, MEM antibiotics of class cephalosporins, sulfonamide and carbapenems.
Identification of antibiotic resistance genes in the isolates (Table 14). Escherichia coli, Pseudomonas and Klebsiella acquire bla SHV, PAGS, PASS, Cnf 1 and hlyC genes, bla TEM gene amplify Pseudomonas and Klebsiella, Fim H gene amplify only Klebsiella, bla CTX-M, CARB KPC and VIM-1 gene were absent in E. coli, Pseudomonas and Klebsiella.
DNA obtain from E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (Figure 2). The amplification was very high in Pseudomonas aeruginosa compare to E. coli and Klebsiella pneumoniae.
Table 9. Percentage Antibiotic Susceptibility of Gram Negative Bacteria (P. luteola, B. cepacia, A. xylosoxidans, B. vietnamiensis, S. odorifera) Isolated from under five Children in General, Teaching and Immanuel Hospital, (Ikot Ekpene, Uyo and Eket).

Nos. of isolated Gram negative bacteria

AP

TET (%)

COT (%)

GEN (%)

CRX (%)

CHL (%)

CTR (%)

CTX (%)

CIP (%)

AMK (%)

VAN (%)

CPZ (%)

MEM (%)

P. luteola (3)

S

3 (100.0)

1 (33.3)

3 (100.0)

0

3 (100.0)

0

0

2 (66.7)

3 (100.0)

3 (100.0)

0

0

R

0

1 (33.3)

0

3 (100.0)

0

3 (100.0)

3 (100.0)

1 (33.3)

0

0

2 (66.7)

3 (100.0)

B. cepacia (3)

S

1 (33.3)

0

2 (66.7)

0

3 (100.0)

1 (33.3)

0

3 (100.0)

3 (100.0)

1 (33.3)

0

0

R

2 (66.7)

2 (66.7)

0

3 (100.0)

0

2 (66.7)

3 (100.0)

0

0

1 (33.3)

3 (100.0)

3 (100.0)

A. xylosoxidans (3)

S

0

1 (33.3)

0

0

3 (100.0)

0

0

3 (100.0)

3 (100.0)

0

0

0

R

3 (100.0)

1 (33.3)

2 (66.7)

3 (100.0)

0

3 (100.0)

3 (100.0)

0

0

2 (66.7)

3 (100.0)

3 (100.0)

B. vietnamiensis (2)

S

0

0

1 (50.0)

0

1 (50.0)

0

0

1 (50.0)

2 (100.0)

0

0

0

R

2 (100.0)

2 (100.0)

0

2 (100.0)

0

2 (100.0)

2 (100.0)

0

0

1 (50.0)

2 (100.0)

2 (100.0)

S. odorifera (1)

S

1 (100.0)

0

1 (100.0)

0

1 (100.0)

0

0

1 (100.0)

1 (100.0)

1 (100.0)

0

0

R

0

1 (100.0)

0

1 (100.0)

0

1 (100.0)

1 (100.0)

0

0

0

1 (100.0)

1 (100.0)

Key: Tetracycline (TET) 10µg, Cotrimoxazole (COT) 25µg, Gentamicin (GEN) 10µg, Amikacin (AMK) 30µg, Vancomycin (VAN) 30µg, Cefuroxime (CRX) 30µg, Ceftriaxone (CTR) 30µg, Cefotaxime (CTX) 30µg, Ceftazidime (CPZ) 30µg, Chloramphenicol (CHL) 10µg, Ciprofloxacin (CIP) 5µg, Meropenem (MEM) 10µg. AP-Antibiotic pattern, S-Susceptibility, R–resistance.
Table 10. Determination of Antibiotic Resistance Pattern of Gram Negative Isolates (E. coli, P. mirabilis, P. aeruginosa) obtained from Sepsis in under five Children in General, Teaching and Immanuel Hospital (Ikot Ekpene, Uyo and Eket).

Gram negative bacteria

Nos. of isolated Gram negative bacteria

Nos. of resistance antibiotic (X)

MARI = X/Y (%)

Antibiotic resistance pattern (ARP)

Antibiotic resistance class (ARC)

Escherichia coli

2

9

9/12 = 0.75 = 75

CRX, CTR, CTX, TET, CIP, COT, VAN, GEN, MEM

Ceph, tetra, fluoro, sulf, glyco, aminogly, carb

2

8

8/12 = 0.7 = 70

CRX, CTX, CPZ, TET, COT, CIP, AMK, MEM

Ceph, sulf, tetra, fluoro, aminogly, carb

6

7

7/12 = 0.6 = 60

CRX, CTR, CTX, CPZ, COT, CIP, MEM

Ceph, sulf, fluoro, carb

5

6

6/12 = 0.5 = 50

CRX, CTX, CPZ, TET, VAN, MEM

Ceph, tetra, glyco, carb

8

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

2

4

4/12 = 0.3 = 30

CTR, CTX, CPZ, MEM

Ceph, carb

25

Proteus mirabilis

2

8

8/12 = 0.7 = 70

CRX, CTR, CTX, CPZ, CIP, COT, CHL, MEM

Ceph, fluoro, sulf, chlo, carb

11

6

6/12 = 0.5 = 50

CTX, CPZ, TET, COT, VAN, MEM

Ceph, tetra, sulf, glyco, carb

5

5

5/12 = 0.4 = 40

CRX, CTX, CPZ, CHL, MEM

Ceph, chlo, carb

1

4

4/12 = 0.3 = 30

CRX, CTX, CPZ, MEM

Ceph, carb

19

Pseudomonas aeruginosa

9

12

12/12 = 1 = 100

CRX, CTR, CTX, CPZ, CIP, TET, COT, CHL, VAN, GEN, AMK, MEM

Ceph, fluoro, tetra, sulf, chlo, glyco, aminogly, carb

1

11

11/12 = 0.9 = 90

CRX, CTR, CTX, CPZ, CIP, TET, COT, CHL, GEN, AMK, MEM

Ceph, fluoro, tetra, sulf, chlo, aminogly, carb

2

8

8/12 = 0.7 = 70

CRX, CTR, CTX, CPZ, TET, COT, CHL, MEM

Ceph, tetra, sulf, chlo, carb

1

7

7/12 = 0.6 = 60

CRX, CTR, CTX, CPZ, TET, COT, MEM

Ceph, tetra, sulf, carb

1

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, CHL, MEM

Ceph, chlo, carb

1

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

15

Table 11. Determination of Antibiotic Resistance Pattern of Gram Negative Isolates (K. pneumoniae, S. ficaria, R. radiobacter) obtained from Sepsis in under five Children in General, Teaching and Immanuel Hospital (Ikot Ekpene, Uyo and Eket).

Gram negative bacteria

Nos. of isolated Gram negative bacteria

Nos. of resistance antibiotic (X)

MARI = X/Y (%)

Antibiotic resistance pattern (ARP)

Antibiotic resistance class (ARC)

Klebsiella pneumoniae

1

10

10/12 = 0.8 = 80

CRX, CTR, CTX, CPZ, CIP, COT, TET, VAN, AMK, MEM

Ceph, fluoro, sulf, tetra, glyco, aminogly, carb

4

9

9/12 = 0.75 = 75

CRX, CTR, CTX, CPZ, TET, COT, CIP, VAN, MEM

Ceph, tetra, sulf, fluoro, glyco, carb

1

8

8/12 = 0.7 = 70

CRX, CTR, CTX, CPZ, TET, COT, CIP, MEM

Ceph, tetra, sulf, fluoro, carb

4

7

7/12 = 0.6 = 60

CTR, CTX, CPZ, TET, COT, CIP, MEM

Ceph, tetra, sulf, fluoro, carb

1

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, CIP, MEM

Ceph, fluoro, carb

1

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

12

Serratia ficaria

2

6

6/12 = 0.5 = 50

CTR, CTX, CPZ, CIP, GEN, MEM

Ceph, fluoro, aminogly, carb

2

5

5/12 = 0.4 = 40

CTR, CTX, CPZ, CHL, MEM

Ceph, chloro, carb

4

4

4/12 = 0.3 = 30

CTR, CTX, CPZ, MEM

Ceph, carb

1

2

2/12 = 0.2 = 20

CTX, MEM

Ceph, carb

9

Rhizobium radiobacter

2

7

7/12 = 0.6 = 60

CRX, CTR, CTX, CPZ, TET, COT, MEM

Ceph, tetra, sulf, carb

3

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, COT, MEM

Ceph, sulf, carb

1

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

2

3

3/12 = 0.3 = 30

CTR, CTX, CHL

Ceph, chloro

8

Key: Tetracycline (TET) 10µg - tetracyclines (tet) or broad spectrum, Cotrimoxazole (COT) 25µg - sulfonamides (sulf), Gentamicin (GEN) 10µg, Amikacin (AMK) 30µg - aminoglycosides (aminogly), Vancomycin (VAN) 30µg - glycopeptides (glyco), Cefuroxime (CRX) 30µg, Ceftriaxone (CTR) 30µg, Cefotaxime (CTX) 30µg, Ceftazidime (CPZ) 30µg - Cephalosporins (ceph), Chloramphenicol (CHL) 10µg - Chloramphenicol (chl), Ciprofloxacin (CIP) 5µg - Fluoroquinolones (quinolones) (fluoro), Meropenem (MEM) 10µg - Carbapenems (carb). MARI - Multiple Antibiotic Resistance Index.
MARI = X/Y where X = number of antibiotics to which test include displayed resistance.
Y = the total number of antibiotics to which the test organism has been evaluated for sensitivity.
Table 12. Determination of Antibiotic Resistance Pattern of Gram Negative Isolates (K. oxytoca, C. violaceum, S. marcescens, E. fergusonii, Pseudomonas luteola) obtained from Sepsis in under five Children in General, Teaching and Immanuel Hospital (Ikot Ekpene, Uyo and Eket).

Gram negative bacteria

Nos. of isolated Gram negative bacteria

Nos. of resistance antibiotic (X)

MARI = X/Y (%)

Antibiotic resistance pattern (ARP)

Antibiotic resistance class (ARC)

Klebsiella oxytoca

2

10

10/12 = 0.8 = 80

CRX, CTR, CTX, CPZ, TET, CIP, COT, VAN, AMK, MEM

Ceph, tetra, fluoro, sulf, glyco, aminogly, carb

4

9

9/12 = 0.75 = 75

CRX, CTR, CTX, CPZ, TET, COT, CIP, GEN, MEM

Ceph, tetra, sulf, fluoro, aminogly, carb

1

8

8/12 = 0.7 = 70

CTR, CTX, CPZ, TET, COT, CIP, GEN, MEM

Ceph, tetra, sulf, fluoro, aminogly, carb

7

Chromo bacterium violaceum

2

7

7/12 = 0.6 = 60

CRX, CTR, CTX, CPZ, VAN, GEN, MEM

Ceph, glyco, aminogly, carb

2

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, COT, MEM

Ceph, sulf, carb

2

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

1

4

4/12 = 0.3 = 30

CTR, CTX, CPZ, MEM

Ceph, carb

7

Serratia marcescens

1

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, COT, MEM

Ceph, sulf, carb

3

5

5/12 = 0.4 = 40

CTR, CTX, CPZ, COT, MEM

Ceph, sulf, carb

1

4

4/12 = 0.3 = 30

CRX, CTR, CTX, MEM

Ceph, carb

5

Escherichia fergusonii

1

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, VAN, MEM

Ceph, glyco, carb

1

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

2

4

4/12 = 0.3 = 30

CTR, CTX, CPZ, MEM

Ceph, carb

4

Pseudomonas luteola

1

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, CIP, MEM

Ceph, fluoro, carb

2

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

3

Table 13. Determination of the Antibiotic Resistance Pattern of Gram Negative Isolates (B. cepacia, A. xylosoxidans, B. vietnamiensis, S. odorifera) obtained from Sepsis in under five Children in General, Teaching and Immanuel Hospital (Ikot Ekpene, Uyo and Eket).

Gram negative bacteria

Nos. of isolated Gram negative bacteria

Nos. of resistance antibiotic (X)

MARI = X/Y (%)

Antibiotic resistance pattern (ARP)

Antibiotic resistance class (ARC)

Burkholderia cepacia

2

7

7/12 = 0.6 = 60

CRX, CTX, CPZ, TET, COT, VAN, MEM

Ceph, tetra, sulf, glyco, carb

1

5

5/12 = 0.4 = 40

CRX, CTR, CTX, CPZ, MEM

Ceph, carb

3

Achromobacter xylosoxidans

1

9

9/12 = 0.75 = 75

CRX, CTR, CTX, CPZ, TET, COT, VAN, GEN, MEM

Ceph, tetra, sulf, glyco, aminogly, carb

1

8

8/12 = 0.7 = 70

CRX, CTR, CTX, CPZ, TET, GEN, VAN, MEM

Ceph, tetra, aminogly, glyco, carb

1

6

6/12 = 0.5 = 50

CRX, CTR, CTX, CPZ, TET, MEM

Ceph, tetra, carb

3

Burkholderia vietnamiensis

1

8

8/12 = 0.7 = 70

CRX, CTR, CTX, CPZ, TET, COT, VAN, MEM

Ceph, tetra, sulf, glycol, carb

1

7

7/12 = 0.6 = 60

CRX, CTR, CTX, CPZ, TET, COT, MEM

Ceph, tetra, sulf, carb

2

Serratia odorifera

1

6

6/12 = 0.5 = 50

CTR, CTX, CRX, CPZ, COT, MEM

Ceph, sulf, carb

1

Total isolated bacteria

123

Key: Tetracycline (TET) 10µg - tetracyclines (tet) or broad spectrum, Cotrimoxazole (COT) 25µg - sulfonamides (sulf), Gentamicin (GEN) 10µg, Amikacin (AMK) 30µg - aminoglycosides (aminogly), Vancomycin (VAN) 30µg – glycopeptides (glyco), Cefuroxime (CRX) 30µg, Ceftriaxone (CTR) 30µg, Cefotaxime (CTX) 30µg, Ceftazidime (CPZ) 30µg - Cephalosporins (ceph), Chloramphenicol (CHL) 10µg - Chloramphenicol (chl), Ciprofloxacin (CIP) 5µg - Fluoroquinolones (quinolones) (fluoro), Meropenem (MEM) 10µg - Carbapenems (carb). MARI - Multiple Antibiotic Resistance Index.
MARI = X/Y where X = number of antibiotics to which test include displayed resistance.
Y = the total number of antibiotics to which the test organism has been evaluated for sensitivity.
Table 14. Identification of Antibiotic Resistance Genes in the Isolates.

Resistant gene

Present of resistant gene in Gram negative bacteria

Absent of resistant gene in Gram negative bacteria

Bla TEM

Pseudomonas and Klebsiella

Escherichia coli

Bla SHV

E. coli, Pseudomonas and Klebsiella

-

Bla CTX-M

-

E. coli, Pseudomonas and Klebsiella

CARB KPC

-

E. coli, Pseudomonas and Klebsiella

VIM-1

-

E. coli, Pseudomonas and Klebsiella

PASS

E. coli, Pseudomonas and Klebsiella

-

PAGS

E. coli, Pseudomonas and Klebsiella

-

FimH

Klebsiella

E. coli, Pseudomonas

Cnf1

E. coli, Pseudomonas and Klebsiella

-

HlyC

E. coli, Pseudomonas and Klebsiella

-

Key: SHV = Sulf-hydryl, TEM = Temoniera, CTX-M = Cefotaximase-munich, cnf1 = Cytotoxic necrotizing factor 1, hlyC = Haemolysin, fimH = Type 1 fimbrae, CARB KPC and VIM-1 = Carbapenemase internal primer genes
Figure 2. DNA obtain from E. coli, P. aeruginosa and K. pneumoniae 1 = E. coli; 2 = P. aeruginosa; 3 = K. pneumoniae.
4. Discussion
Sepsis is a major cause of morbidity and mortality in neonates globally ranging from 30-50% in developing countries (Klick and Guins, 2021) . Gram negative bacteria play an important role in causing nosocomial and non-nosocomial infections (Rasool et al., 2019; Miranda et al., 2024) . Death rate in neonates comprise of 41% (3.6 million) in children below 5 years. About 1 million of these deaths are linked with infectious diseases like pneumonia, neonatal sepsis and meningitis (Getabelew et al., 2018) . This study has demonstrated the continued role of Septicaemia in under-five children in three tertiary hospitals, Akwa Ibom State, Nigeria.
The prevalence of Gram negative bacteria was found to be 68.3% which is comparable to different studies ranging from 18 to 95% (Miranda et al., 2024; Godfrey et al., 2022; Habyarimana et al., 2021; Almohammady et al., 2020; Adeodokun et al., 2020 in Nigeria ; Droz et al., 2019; Peterside et al., 2015 in Port Harcourt Nigeria; Shehab et al., 2015; Joshi et al., 2000) , the high prevalence could be attributed to: (1) antibiotic resistance, which is due to the overuse of broad-spectrum antibiotics (2) environmental factors like medical devices or gastrointestinal tract (3) lifestyle modifications: changing antibiotic usage patterns and lifestyle modification can change the microorganisms that cause sepsis. Contrary to this work, Aletayeb et al., (2011) in Nigeria, reported a very low positive blood culture of neonates 4.1% which they attributed it to antibiotic administration in mother or neonate, difficulty in sampling, blood culture technique (Bansal et al., 2004) or sepsis due to anaerobic, viral or fungal pathogens (Agnihotri et al., 2004; Raha et al., 2014) and misdiagnosis due to some similarities between the clinical signs of sepsis with other diseases like metabolic disorders (Lund et al., 2002; Vinodkumar et al., 2008) . In other words, hygienic conditions and use of antibiotics (Giannoni et al., 2018) , sources and numbers of clinical samples collected, type of infections, types of patients and probably geographical differences might be the cause of variation in the prevalence of Gram negative bacteria.
The most frequent isolated Gram negative bacteria among the three hospitals were, E. coli, P. mirabilis, K. oxytoca, S. ficaria, R. radiobacter, S. marcescens and A. xylosoxidans ranging from 1.7% - 23.3%. A. xylosoxidans, though present in the three hospitals under this study, were the least with 1(1.7%). This is in accordance with a study by Laxman and Roja, (2022) , describing A. xylosoxidans as an unusual pathogen. Among the three hospitals, E. coli had the highest prevalence 13.9%, which is comparable to Peterside et al., (2015) ; 16.5%, Godfrey et al., (2022) ; 12.3%, and Oyekale et al., (2022) ; 29.4%. This is due to the fact that E. coli is a gastrointestinal tract infection that would likely be transmitted perinatally. It might also be due to the proximity of the anal opening to the urethra as the E. coli resides as commensals in the gastrointestinal tract (Kebede et al., 2022) . In contrast, studies in Nigeria by Aletayeb et al., (2011) ; Arowosegbe et al., (2017) and Shobowale et al., (2017) shows K. pneumoniae as the most predominant isolates. This was attributed to the fact that K. pneumoniae can reside in the hospital environment, the gastrointestinal tract, the birth canal and on the surfaces of medical devices. However, difference in bacterial isolates can be due to epidemiological and geographical distribution of bacteria and seasonal variation.
K. Pneumoniae and P. aeruginosa, among the highest causative Gram negative bacteraemia in this study were absent in one of the hospitals (Immanuel hospital, Eket). This could be attributed to sources and numbers of clinical samples collected and geographical differences.
In sepsis, multidrug resistant organisms are crucially involved in causing high mortality in neonates as compared to non-multidrug resistant bacteria (Yusef et al., 2018) . However Nigeria has been reported as one of the countries with high resistance to antibiotics by Gram negative organisms (Popescu et al., 2020) . We found Escherichia spp, Proteus mirabilis, Klebsiella pneumoniae, Serratia spp, Rhizobium radiobacter, Chromobacterium violaceum, Pseudomonas luteola, Burkholderia spp, and Achromobacter xylosoxidans 100% susceptible to amikacin, which shows a high efficacy of this drug in treatment of neonatal sepsis. This is similar to a study by Tesini (2022) . However, 100% C. violaceum susceptibility to CIP reported in this work, confirms the findings of Jiang et al., (2024) who revealed the efficacy in treatment of children with CIP 62.5%. We report Klebsiella pneumoniae 100% resistance to CTX, CPZ and MEM, similar to a study by Nwadioha et al., (2011) and Onipede et al., (2009) , who revealed 64% K. pneumoniae resistance to CTX. This could be attributed to the fact that, K. pneumoniae lives in both the soil and the gut, which are both hotspots for the intergenus transfer of antibiotic resistance (Mukherjee et al., 2021) .
In this study, all the organisms were 100% resistance to CTX among all the cephalosporins, which is in agreement with findings of Bai et al., (2021) . In contrast, Ahmad et al., (2021) reports 50% resistance to CTX. E. coli, P. mirabilis, P. aeruginosa, K. pneumoniae, Klebsiella spp., A. xylosoxidans and Burkholderia spp. showed 100% resistance to CTX, CPZ, MEM, which is comparable with Ogbolu et al., (2019) ; Obadare et al., (2021) ; Obaro et al., (2022) ; Obe et al., (2023) findings. The high resistance rate against many antibiotics could be attributed to; inappropriate and overuse of antibiotics, sometimes clinicians initiating antibiotic therapy before performing blood culture, might have accounted for the high resistance rates detected in our study. It can also be due to the fact that, these organisms develop antibiotic resistance through efflux pumps (over expression of proton transporter AcrAB and protein ToIC), beta-lactamase production and outer membrane alteration mechanisms.
The choice of first-line antibiotic for the initiation of treatment in cases of neonatal sepsis is a challenge to a clinician. It is further complicated when it is suspected to be caused by drug-resistant bacteria. Βeta-lactam antibiotics are used widely worldwide against infections caused by Gram-negative bacteria. Resistance is known to be due to various mechanisms among which production of extended-spectrum β-lactamases (ESBLs) was reported (Roy et al., 2013; Vijayakanthi et al., 2013; Datta et al., 2015; Ravikant et al., 2016) . Presence of ESBLs has been reported among bacterial isolates from the neonates (Bora and Ahmed, 2012; Roy et al., 2013; Vijayakanthi et al., 2013; Datta et al., 2015; Devi et al., 2018) . The multiplex PCR amplification assay was used in this study to identify the blaTEM, blaSHV, blaCTX-M-1, CARB KPC, VIM-1, PASS, PAGs, Fim H, Cnf 1 and Hlyc genes in E. coli, Klebsiella and Pseudomonas clinical isolates. Fim H gene was found to amplify only klebsiella but absence in E. coli and pseudomonas. Cnf 1 gene amplify E. coli, Klebsiella and Pseudomonas but showed low base pair for the three isolates, which could be due to in appropriate designing of the primer. Though there is a low base pair for these isolates, there is still gene present in this isolates, revealing the presence of resistance gene in the organisms. PAGs and PASS gene showed a high quality fine peak in P. aeruginosa. This might be due to the fact that the gene was specific for P. aeruginosa. However, these genes were seen as the most prevalent contrary to a study by Azab et al., 2021 and a study in Iraq and USA which shows blaCTX-M as the most prevalent kind of Beta-lactamase (Ahmad Hamad and Khadija, 2019; Jorgensen et al., 2010) . A study in Burkina Faso and United State also revealed blaCTX-M as the most predominant gene (Doi et al., 2013; Ouedraogo et al., 2016) . This is attributed to geographical variances between countries. BlaTEM gene, which was known to be the specific gene for E. coli has been reported and confirmed by other studies (Devrim et al., 2018; Ahmad Hamad and Khadija 2019; Uyanga et al., 2020; Al-Tahish et al., 2024) . This is contrary to our work in which blaTEM gene did not amplify E. coli. A study in Saudi Arabia and Egypt also showed blaTEM gene was not detected in E. coli (Ojdana et al., 2014) . This means the resistant gene did not express in E. coli and the organisms do not have the resistant gene or the gene region that was used to design the primer was not presence in the organism. In our study, blaCTX-M was the least common extended-spectrum beta-lactamase ESBLs contrary to a study by Al-Tahish et al., 2024 that shows blaSHV as the least common ESBLs.
5. Conclusions
A high prevalence of resistance to almost all the first line antibiotics by Gram negative organisms, mostly cefotaxime causing sepsis in under-five children has been revealed in this study. Although antibiotics can control infections speedily and prevent sepsis in under-five children to some extent, their use could also easily induce a diversity of highly resistant strains. Pediatricians should strictly control the conditions under which antibiotics are applied and replace them at some point (according to the specifics of a given situation) to reduce the occurrence of drug resistance and improve drug efficacy. However, 100% susceptibility of all Gram negative organisms to amikacin in this study shows a high efficacy of this drug in treatment of sepsis in under-five children. This study indicates that Gram negative species especially, E. coli, P. mirabilis, P. aeruginosa, K. pneumoniae, S. ficaria, R. radiobacter continue to be the most predominant causative organisms in neonatal sepsis. All emergency departments should have a screening tool and sepsis bundle to aid early identification of the septic child with timely management and appropriate acceleration. It is recommended that further studies should be carried out with a larger molecular analysis and population size.
Abbreviations

TEM

Temoniera

SHV

Sulf-hydryl

PAGs

Pseudomonas Aeruginosa Genes

PASS

Pseudomonas Aeruginosa Subspecie

Cnf1

Citotoxic Necrotizing Factor 1

hlyc

haemolysin

Fim H

Type1 Fimbrae

CDC

Center for Disease and Control

MDR

Multidrug Resistance

MAR

Multiple Antibiotics Resistance

CTX-M

Cifotàximase Munich

SSI

Surgical Site Infection

VAP

Ventilator Associated Pneumonia

CLABSI

Central Line Associated Blood Stream Infection

MRSA

Methicillin Resistant Staphylococcus aureus

WHO

World Health Organization

LMICs

Low and Middle Income Countries

NPC

National Population Commission

CLSI

Clinical Laboratory Standards Institute

ARC

Antibiotic Resistant Class

ARP

Antibiotic Resistant Pattern

TET

Tetracycline

CoT

Cotrimoxazole

GEN

Gentamicin

CRX

Cefuroxime

CHL

Chloramphenicol

CTR

Ceftriaxone

CTX

Cefotaxime

CIP

Ciprofloxacin

AMK

Amikacin

VAN

Vancomycin

CPZ

Ceftazidime

MEM

Meropenem

DNA

Deoxyribonucleic Acid

OD

Optical Density

EDTA

Ethylinediamine-Tetraacetic Acid

PCR

Polymerase Chain Reaction

S

Susceptibility

AP

Antibiotic Pattern

R

Resistance

ESBLS

Extended Spectrum Beta Lactamases

Acknowledgments
For their invaluable collaboration and support, we sincerely appreciate the laboratory staff at the Genomic Training Center Uyo, Akwa Ibom State University, Ritman University Ikot Ekpene, General Hospital Ikot Ekpene, Uyo Teaching Hospital, and Immanuel Hospital Eket, Akwa Ibom State.
Author Contributions
Christopher Mary: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing
Umoh Jarlath: Conceptualization, Data curation, Project administration, Supervision, Validation, Visualization, Writing–review & editing
Owowo Etanguno: Conceptualization, Data curation, Project administration, Supervision, Validation, Visualization, Writing–review & editing
Bassey Maria: Conceptualization, Data curation, Project administration, Supervision, Validation, Visualization, Writing–review & editing
Nyoyoko Veronica: Conceptualization, Data curation, Project administration, Validation, Visualization, Writing–review & editing
Funding
No funding of research but as part of employment, manuscript writing was done by the first author, editing, approval or decision to publish was done by other authors.
Conflicts of Interest
The authors declare no conflicts of interest.
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    Mary, C., Jarlath, U., Etanguno, O., Maria, B., Veronica, N. (2025). Molecular Characterization of Gram Negative Bacteria Involved in Sepsis Among Under Five Children in Akwa Ibom State Nigeria. International Journal of Microbiology and Biotechnology, 10(3), 111-130. https://doi.org/10.11648/j.ijmb.20251003.15

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    Mary, C.; Jarlath, U.; Etanguno, O.; Maria, B.; Veronica, N. Molecular Characterization of Gram Negative Bacteria Involved in Sepsis Among Under Five Children in Akwa Ibom State Nigeria. Int. J. Microbiol. Biotechnol. 2025, 10(3), 111-130. doi: 10.11648/j.ijmb.20251003.15

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    AMA Style

    Mary C, Jarlath U, Etanguno O, Maria B, Veronica N. Molecular Characterization of Gram Negative Bacteria Involved in Sepsis Among Under Five Children in Akwa Ibom State Nigeria. Int J Microbiol Biotechnol. 2025;10(3):111-130. doi: 10.11648/j.ijmb.20251003.15

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  • @article{10.11648/j.ijmb.20251003.15,
      author = {Christopher Mary and Umoh Jarlath and Owowo Etanguno and Bassey Maria and Nyoyoko Veronica},
      title = {Molecular Characterization of Gram Negative Bacteria Involved in Sepsis Among Under Five Children in Akwa Ibom State Nigeria
    },
      journal = {International Journal of Microbiology and Biotechnology},
      volume = {10},
      number = {3},
      pages = {111-130},
      doi = {10.11648/j.ijmb.20251003.15},
      url = {https://doi.org/10.11648/j.ijmb.20251003.15},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijmb.20251003.15},
      abstract = {Sepsis is a life threatening medical emergency, Gram negative bacteria the principal causes of sepsis are seen in higher proportions among pediatrics populations and are mostly antibiotic resistance organisms. This study was carried out to determine antibiotic resistance of Gram negative bacteria, antibiotic resistance genes involve in sepsis among under five children in Akwa Ibom State, Nigeria. A hospital-based descriptive observational study of neonates with or without clinical features of sepsis. The subjects were children seen in General Hospital Ikot Ekpene, University Teaching Hospital Uyo and Immanuel Hospital Eket. A total of 180 children were sampled (60 from each hospital). A two milliliters (2 ml) sterile syringe with a 23gauge needle was used to collect blood sample aseptically from the vein of the arm of the child, inoculated on thioglycollate broth and subculture on MaConkey, blood and chocolate agar. Gram staining, biochemical characterization, antimicrobial susceptibility and resistance of Gram negative bacteria, their resistance genes were done. Of the 180 children, 123 tested positive for bacterial infections. Escherichia coli 25(13.9%), Proteus mirabilis 19(10.6%), Pseudomonas aeruginosa 15(8.3%), Klebsiella pneumoniae 12(6.7%), Serratia ficaria 9(5.0%), Rhizobium radiobacter 8(4.4%), Klebsiella oxytoca 7(3.9%), Chromobacterium violaceum 7(3.9%), Serratia marcescens 5(2.8%), Escherichia fergusonii 4(2.2%), Pseudomonas luteola 3(1.7%), Burkholderia cepacia 3(1.7%), Achromobacter xylosoxidans 3(1.7%), Burkholderia vietnamiensis 2(1.1%) and Serratia odorifera 1(0.6%). Pseudomonas aeruginosa was resistance to the 12 antibiotic used 12(100%), Three isolates were finally selected for molecular analysis, E. coli, P. aeruginosa, K. pneumoniae acquire blaSHV, PAGS, PASS, Cnf1 and hlyC genes, blaTEM amplify Pseudomonas and Klebsiella, FimH amplify only Klebsiella. Gram negative bacteria develop antibiotic resistance which poses a significant challenge in treating infections caused by this organism emphasizing the importance of responsible antibiotic use to mitigate further development of resistance.
    },
     year = {2025}
    }
    

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  • TY  - JOUR
    T1  - Molecular Characterization of Gram Negative Bacteria Involved in Sepsis Among Under Five Children in Akwa Ibom State Nigeria
    
    AU  - Christopher Mary
    AU  - Umoh Jarlath
    AU  - Owowo Etanguno
    AU  - Bassey Maria
    AU  - Nyoyoko Veronica
    Y1  - 2025/09/11
    PY  - 2025
    N1  - https://doi.org/10.11648/j.ijmb.20251003.15
    DO  - 10.11648/j.ijmb.20251003.15
    T2  - International Journal of Microbiology and Biotechnology
    JF  - International Journal of Microbiology and Biotechnology
    JO  - International Journal of Microbiology and Biotechnology
    SP  - 111
    EP  - 130
    PB  - Science Publishing Group
    SN  - 2578-9686
    UR  - https://doi.org/10.11648/j.ijmb.20251003.15
    AB  - Sepsis is a life threatening medical emergency, Gram negative bacteria the principal causes of sepsis are seen in higher proportions among pediatrics populations and are mostly antibiotic resistance organisms. This study was carried out to determine antibiotic resistance of Gram negative bacteria, antibiotic resistance genes involve in sepsis among under five children in Akwa Ibom State, Nigeria. A hospital-based descriptive observational study of neonates with or without clinical features of sepsis. The subjects were children seen in General Hospital Ikot Ekpene, University Teaching Hospital Uyo and Immanuel Hospital Eket. A total of 180 children were sampled (60 from each hospital). A two milliliters (2 ml) sterile syringe with a 23gauge needle was used to collect blood sample aseptically from the vein of the arm of the child, inoculated on thioglycollate broth and subculture on MaConkey, blood and chocolate agar. Gram staining, biochemical characterization, antimicrobial susceptibility and resistance of Gram negative bacteria, their resistance genes were done. Of the 180 children, 123 tested positive for bacterial infections. Escherichia coli 25(13.9%), Proteus mirabilis 19(10.6%), Pseudomonas aeruginosa 15(8.3%), Klebsiella pneumoniae 12(6.7%), Serratia ficaria 9(5.0%), Rhizobium radiobacter 8(4.4%), Klebsiella oxytoca 7(3.9%), Chromobacterium violaceum 7(3.9%), Serratia marcescens 5(2.8%), Escherichia fergusonii 4(2.2%), Pseudomonas luteola 3(1.7%), Burkholderia cepacia 3(1.7%), Achromobacter xylosoxidans 3(1.7%), Burkholderia vietnamiensis 2(1.1%) and Serratia odorifera 1(0.6%). Pseudomonas aeruginosa was resistance to the 12 antibiotic used 12(100%), Three isolates were finally selected for molecular analysis, E. coli, P. aeruginosa, K. pneumoniae acquire blaSHV, PAGS, PASS, Cnf1 and hlyC genes, blaTEM amplify Pseudomonas and Klebsiella, FimH amplify only Klebsiella. Gram negative bacteria develop antibiotic resistance which poses a significant challenge in treating infections caused by this organism emphasizing the importance of responsible antibiotic use to mitigate further development of resistance.
    
    VL  - 10
    IS  - 3
    ER  - 

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Author Information
  • Department of Microbiology, Faculty of Biological Sciences, Akwa Ibom State University, Ikot Akpaden, Nigeria

  • Department of Microbiology, Faculty of Biological Sciences, Akwa Ibom State University, Ikot Akpaden, Nigeria

  • Department of Microbiology, Faculty of Biological Sciences, Akwa Ibom State University, Ikot Akpaden, Nigeria

  • Department of Microbiology, Faculty of Biological Sciences, Akwa Ibom State University, Ikot Akpaden, Nigeria

  • Department of Biological Sciences, Topfaith University, Mkpatak, Nigeria

  • Abstract
  • Keywords
  • Document Sections

    1. 1. Introduction
    2. 2. Materials and Methods
    3. 3. Results
    4. 4. Discussion
    5. 5. Conclusions
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  • Abbreviations
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  • Author Contributions
  • Funding
  • Conflicts of Interest
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