Original Article

Molecular Characterization of Hospital-Acquired Carbapenem-Resistant Gram-Negative Pathogenic Bacteria From a Tertiary Hospital in Lagos State, Nigeria

Please cite this article as follows: Eze IF, Adeoye-Isijola M, Adefisoye MA, Olajuyigbe OO. Molecular characterization of hospital-acquired carbapenem-resistant gram-negative pathogenic bacteria from a Tertiary hospital in Lagos State, Nigeria. Avicenna J Clin Microbiol Infect. 2025;12(2):56-60. doi:10.34172/ajcmi.3619

Introduction

Antibiotic resistance is a major challenge in hospital-acquired bacterial infections, and this has led to a failure in the treatment of such infections, especially in developing countries (1). Carbapenems are reserve antibiotics used for the treatment of multidrug-resistant, life-threatening diseases. Carbapenem-resistant gram-negative bacteria are a major challenge in antibiotic resistance globally due to their innate ability to cause life-threatening diseases. In addition, they are among the top three drug-resistant bacteria in dire need of new antibiotics (2). Carbapenem-resistant Enterobacterales destroy antibiotics that were initially highly active against gram-negative organisms, limiting antibiotic options to new drugs with reduced effectiveness and older ones with high toxicity. Carbapenem resistance in gram-negative bacteria remains an emerging and significant public health threat, especially in low-income and medium-income countries, including Nigeria (3). In Nigeria, there is a paucity of records on carbapenem resistance in hospital-acquired gram-negative bacteria; antibiotic prescription is high, with only 1/2 of prescriptions based on proper therapeutic reasons and one-third of prescriptions having a broad-spectrum antibiotic (4). The molecular characterization of these carbapenem-resistant gram-negative bacteria strains is essential for monitoring and controlling their disease outbreak. Therefore, this study aims to identify specific strains of hospital-acquired carbapenem-resistant gram-negative bacteria using molecular techniques.

Materials and Methods

Study Design and Setting

This study was performed at the Lagos State University Teaching hospital, Ikeja, Lagos, from February 2023 to April 2024. Lagos State University Teaching Hospital is a state-owned tertiary hospital located in southwestern Nigeria and serves as a referral centre for patients in Lagos and its environs. The molecular characterization of isolated bacteria was performed at the Nigeria Institute of Medical Research, Lagos.

Study Criteria

Inclusion Criteria

Gram-negative bacterial isolates obtained from the urine and wound cultures of patients admitted to the hospital for 48 hours or more, showing clinical symptoms for nosocomial infections and having no evidence of this infection on admission, were included in this study.

Exclusion Criteria

Bacterial isolates from the cultures of patients admitted within less than 48 hours or patients not demonstrating clinical symptoms for nosocomial infections were not included in this study.

Study Population

Overall, 162 gram-negative bacteria were isolated from the wound and urine cultures of patients suspected of having hospital-acquired infections.

Collection and Identification of Bacterial Isolates

One hundred and sixty-two non-duplicate clinical isolates of gram-negative bacteria from the cultures of urine and wound samples of inpatients submitted to the Medical Microbiology and Parasitology Unit Laboratory of Lagos State University Teaching Hospital were collected in Nutrient Agar broth. Then, they were phenotypically identified using selective/differential diagnostic media and biochemical tests according to standard microbiological protocols (5).

Carbapenem Susceptibility Screening Tests

Phenotypic screening for carbapenem resistance was performed by the Kirby-Bauer disc diffusion method using meropenem (10 µg) and imipenem (10 µg) discs. Standard inoculum, standardized by comparing it with 0.5 McFarland standards, was uniformly spread on the entire surface area of sterilized Mueller-Hinton agar plates using a cotton swab stick. The carbapenem discs were placed on the surface of the inoculated agar plates using sterile forceps, and the plates were incubated at 37 ^ºC for 24 hours. The zones of inhibition were measured, and the results were interpreted based on standard reference values of the Clinical and Laboratory Standards Institute guidelines (6). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains for the Enterobacteriaceae and non-Enterobacteriaceae, respectively. The isolates were recorded as carbapenem-resistant if they showed resistance to both meropenem and imipenem (7). Pure colonies of the selected bacterial isolates were stored in 15% glycerol stock at -20 ^ºC for further studies.

Molecular Characterization of the Isolates of Carbapenem-Resistant Gram-Negative Bacteria

Molecular characterization of target bacterial isolates was performed based on 16S ribosomal RNA conserved gene sequences. The DNA of carbapenem-resistant bacteria isolates was extracted using the Nigerian Institute of Medical Research genomic DNA extraction kit (a spin column-based DNA purification kit for the isolation of genomic DNA from bacterial cells). The extracted DNA was amplified using a thermocycler (Thermo Fisher Scientific Inc.) for the polymerase chain reaction (PCR). The FIREPol Master Mix (Solis BioDyne) ready to load is a concentrated, ready-to-use solution containing all reagents required for PCR (except DNA template, primers, and water), additional compounds for direct loading onto agarose gel, and two tracking dyes (blue and yellow) to monitor progress during electrophoresis. The 16S ribosomal RNA (16S rRNA) sequence of the selected bacterial isolates was amplified using 1.4 kb bacterial primers 16S rRNA27F (5¹-AGAGTTTGATCCTGGCTCAG-3¹) and 16S rRNA1329R (5¹-GGTTACCTTGTTACGACTT-3¹) in a 20 μL PCR reaction mixture. Based on the manufacturer’s instructions, a negative control made up of the reaction mixture with water instead of DNA was included in each run. The 16S rRNA PCR 20 µL reaction mix cocktail consists of master mix (4 µL), forward primer (0.6 µL), reverse primer (0.6 µL), nuclease-free water (12.8 µL), and bacterial DNA (2 µL). The DNA mix was placed into the thermal cycler, and the PCR cycling protocol is presented in Table 1. The amplified fragment sizes were confirmed and visualized using a 1 kb DNA ladder via gel electrophoresis on 1.5% (w/v) agarose gel in 0.5 x TAE buffer at a constant voltage of 100 V for 30 minutes.

The amplicons were sequenced using the ABI 3500XL Genetic Analyzer (Thermo Fisher Scientific Inc.), and the retrieved nucleotide sequence data were analyzed using Chromas bioinformatics software. The resulting bacterial nucleotide sequences underwent further analysis by the Basic Local Alignment Search Tool on the National Centre for Biotechnology Information website. The nucleotide sequences were submitted to GenBank, and accession numbers were assigned to them.

Results

In general, 106 and 56 isolates of gram-negative bacteria were obtained from urine and wound cultures, respectively. Among the 162 gram-negative isolates, 13 (8%) were carbapenem-resistant (resistant to both meropenem and imipenem). The carbapenem-resistant gram-negative bacteria were from urine cultures, except for Klebsiella pneumoniae, which was from the wound. The molecularly characterized carbapenem-resistant nosocomial pathogens are E. coli, K. pneumoniae, P. aeruginosa, Enterobacter hormaechei, Alcaligenes faecalis, and Providencia stuartii.

The carbapenem-resistant gram-negative bacteria nucleotide sequences (10/13) showed significant gene sequence similarity. The bacterial strain NAF had the highest gene similarity index (99.38%) to E. coli. Bacterial strains Y, X₂, J₃, I₃, C₂, B₂, T₂, Z₂, and C had 99%, 98.85%, 98.71%, 98.53%, 98.26%, 97.80%, 97.40%, 97.18%, and 90.49% gene similarity indices to K. pneumoniae, P. aeruginosa, E. hormaechei, E. hormaechei, A. faecalis, P. stuartii, P. aeruginosa, E. coli, and E. coli, respectively. The strains of hospital-acquired gram-negative bacterial pathogens, identified by molecular characterization, had percentage identity ranging from 90.49% to 99.38% with GenBank accession numbers for their nucleotide sequences from PP808942 to PP808951. Table 2 provides the sequence similarity value of bacterial strains and their accession numbers from the National Centre for Biotechnology Information GenBank.

Discussion

Hospital-acquired carbapenem-resistant gram-negative bacteria strains were molecularly characterized. The susceptibility tests performed in this research for carbapenem resistance were strictly based on the resistance of bacterial isolates to both imipenem and meropenem (7,8). Previous studies demonstrated that carbapenem-resistant Enterobacterales are generally resistant to cephalosporins, penicillins, and other antibiotic classes (8,9). Providencia stuartii is not a common opportunistic organism but has been implicated in healthcare-associated infections, which is in line with the results of this study. Some studies reported that hospital-acquired pathogens cause polymicrobial catheter-associated urinary tract infections (10) and outbreaks in nursing homes and intensive care units (11). However, in Nigeria, there is a lack of information on hospital-acquired carbapenem-resistant P. Stuartii infections. Carbapenem-resistant P. Stuartii from urine was identified in this study, confirming the presence of extensively drug-resistant strains of these bacteria in Lagos State. Extensively drug-resistant P. stuartii is a major problem in North Africa and the Southern Mediterranean, as reported in another study on P. stuartii infections(12). Carbapenem-resistant E. hormaechei is a clinically important hospital-acquired pathogen detected in this study, which conforms to the results of other studies, mainly in Asia, where carbapenem resistance in E. hormaechei had been reported (13,14). Nonetheless, there is a lack of information on the molecular epidemiology of carbapenem-resistant hospital-acquired E. hormaechei infections in Nigeria; this could be due to the paucity of research in this area and lack of appropriate molecular diagnostic tools. A multidrug-resistant P. stuartii strain associated with urine nosocomial infection was identified in a similar study conducted in Greece (15).

E. coli was the most prevalent carbapenem-resistant hospital-acquired pathogen in this study, which corroborates the findings of other similar works on carbapenem-resistant bacteria (16-18). The hospital-acquired carbapenem-resistant A. faecalis strain demonstrated the hospital transmission of these strains in the population of this study, which is comparable to the results of a similar study in Asia, which showed extensive drug resistance by A. faecalis (19).In Nigeria, there is a lack of information on carbapenem resistance by A. faecalis, E. hormaechei, and P. stuartii in hospital-acquired infections (20), implying that these pathogens are emerging hospital-acquired pathogens in Lagos State, Nigeria.

Conclusion

Carbapenem-resistant Enterobacteriaceae are a global health threat in antibiotic therapy, resulting in the explosion of hospital-acquired infections. The most common molecularly characterized gram-negative hospital-acquired carbapenem-resistant bacteria in this study were E. coli, P. stuartii, A. faecalis, and E. hormaechei, respectively. These molecularly characterized gram-negative carbapenem-resistant hospital-acquired bacteria pathogens are emerging nosocomial pathogens in Lagos State, Nigeria. This study has provided knowledge of phenotypes and genotypes of carbapenem-resistant gram-negative nosocomial Enterobacterales in the study population, which will further aid in understanding emerging resistance determinants. The clinical monitoring of the changing epidemiology of these carbapenem-resistant gram-negative bacteria acquired from the hospital environment is essential in preventing disease outbreaks by these bacteria. This could be achieved by effective antibiotic stewardship programs and improved infection control strategies in the hospital.

Acknowledgments

We acknowledge the staff of the Molecular Biology and Biotechnology Department, Nigeria Institute of Medical Research, Lagos, for their assistance during the molecular characterization of bacterial isolates.

Competing Interests

The authors declare that they have no conflict of interests.

Ethical Approval

This study was approved by the Ethics Committee of Lagos State University Teaching Hospital (ethical No. LREC/06/10/2012). Informed consent was obtained from the participants.

Funding

This study received no financial support.

References

1. Sharma A, Singh A, Dar MA, Kaur RJ, Charan J, Iskandar K. Menace of antimicrobial resistance in LMICs: current surveillance practices and control measures to tackle hostility. J Infect Public Health 2022; 15(2):172-81. doi: 10.1016/j.jiph.2021.12.008 [Crossref] [ Google Scholar]
2. Ma J, Song X, Li M, Yu Z, Cheng W, Yu Z. Global spread of carbapenem-resistant Enterobacteriaceae: epidemiological features, resistance mechanisms, detection and therapy. Microbiol Res 2023; 266:127249. doi: 10.1016/j.micres.2022.127249 [Crossref] [ Google Scholar]
3. Tula MY, Enabulele OI, Ophori EA, Aziegbemhin AS, Iyoha O, Filgona J. A systematic review of the current status of carbapenem resistance in Nigeria: its public health implication for national intervention. Niger Postgrad Med J 2023; 30(1):1-11. doi: 10.4103/npmj.npmj_240_22 [Crossref] [ Google Scholar]
4. Oduyebo OO, Olayinka AT, Iregbu KC, Versporten A, Goossens H, Nwajiobi-Princewill PI. A point prevalence survey of antimicrobial prescribing in four Nigerian Tertiary Hospitals. Ann Trop Pathol 2017; 8(1):42-6. [ Google Scholar]
5. Clinical & Laboratory Standards Institute (CLSI). M35-A2: Abbreviated Identification of Bacteria and Yeast; Approved Guideline. 2nd ed. CLSI; 2008.
6. Clinical & Laboratory Standards Institute (CLSI). M100: Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI; 2020.
7. Ugah UI, Udeani TK. Laboratory survey of carbapenem-resistant Enterobacterales isolates in selected tertiary hospitals in south-eastern Nigeria. Int J Med Res Health Sci 2022; 11(8):1-7. [ Google Scholar]
8. Zubair KO, Iregbu KC. Resistance pattern and detection of metallo-beta-lactamase genes in clinical isolates of Pseudomonas aeruginosa in a central Nigeria tertiary hospital. Niger J Clin Pract 2018; 21(2):176-82. doi: 10.4103/njcp.njcp_229_17 [Crossref] [ Google Scholar]
9. Mehrad B, Clark NM, Zhanel GG, Lynch JP 3rd. Antimicrobial resistance in hospital-acquired gram-negative bacterial infections. Chest 2015; 147(5):1413-21. doi: 10.1378/chest.14-2171 [Crossref] [ Google Scholar]
10. Johnson AO, Forsyth V, Smith SN, Learman BS, Brauer AL, White AN. Transposon insertion site sequencing of Providencia stuartii: essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements. mSphere 2020; 5(3):e00412-20. doi: 10.1128/mSphere.00412-20 [Crossref] [ Google Scholar]
11. Liu J, Wang R, Fang M. Clinical and drug resistance characteristics of Providencia stuartii infections in 76 patients. J Int Med Res 2020; 48(10):300060520962296. doi: 10.1177/0300060520962296 [Crossref] [ Google Scholar]
12. Oikonomou O, Liakopoulos A, Phee LM, Betts J, Mevius D, Wareham DW. Providencia stuartii isolates from Greece: co-carriage of cephalosporin (blaSHV-5, blaVEB-1), carbapenem (blaVIM-1), and aminoglycoside (rmtB) resistance determinants by a multidrug-resistant outbreak clone. Microb Drug Resist 2016; 22(5):379-86. doi: 10.1089/mdr.2015.0215 [Crossref] [ Google Scholar]
13. Ding M, Shi J, Ud Din A, Liu Y, Zhang F, Yan X. Co-infections of two carbapenemase-producing Enterobacter hormaechei clinical strains isolated from the same diabetes individual in China. J Med Microbiol 2021; 70(3):001316. doi: 10.1099/jmm.0.001316 [Crossref] [ Google Scholar]
14. Ghazawi A, Anes F, Mouftah S, Elbediwi M, Baig A, Alketbi M. Genomic study of high-risk clones of Enterobacter hormaechei collected from tertiary hospitals in the United Arab Emirates. Antibiotics (Basel) 2024; 13(7):592. doi: 10.3390/antibiotics13070592 [Crossref] [ Google Scholar]
15. Liakopoulos A, Oikonomou O, Wareham DW. Draft genome sequence of Providencia stuartii PS71, a multidrug-resistant strain associated with nosocomial infections in Greece. Genome Announc 2017; 5(12):e00056-17. doi: 10.1128/genomeA.00056-17 [Crossref] [ Google Scholar]
16. Olowo-Okere A, Ibrahim YK, Olayinka BO, Ehinmidu JO, Mohammed Y, Nabti LZ. Phenotypic and genotypic characterization of clinical carbapenem-resistant Enterobacteriaceae isolates from Sokoto, northwest Nigeria. New Microbes New Infect 2020; 37:100727. doi: 10.1016/j.nmni.2020.100727 [Crossref] [ Google Scholar]
17. Olalekan A, Onwugamba F, Iwalokun B, Mellmann A, Becker K, Schaumburg F. High proportion of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae among extended-spectrum β-lactamase-producers in Nigerian hospitals. J Glob Antimicrob Resist 2020; 21:8-12. doi: 10.1016/j.jgar.2019.09.007 [Crossref] [ Google Scholar]
18. Maduakor UC, Eleazar CI, Mba CG, Obodochukwu CC, Eberechukwu CL, Ogu CO. Metallo-beta-lactamase producing isolates of Escherichia coli and Klebsiella pneumoniae and their resistance profiles in Enugu, Nigeria: a threat to public health. J Adv Microbiol 2024; 24(2):11-9. doi: 10.9734/jamb/2024/v24i2791 [Crossref] [ Google Scholar]
19. Huang C. Extensively drug-resistant Alcaligenes faecalis infection. BMC Infect Dis 2020; 20(1):833. doi: 10.1186/s12879-020-05557-8 [Crossref] [ Google Scholar]
20. Tickler IA, Shettima SA, Dela Cruz CM, Le VM, Dewell S, Sumner J. Characterization of carbapenem-resistant gram-negative bacterial isolates from Nigeria by whole genome sequencing. Diagn Microbiol Infect Dis 2021; 101(1):115422. doi: 10.1016/j.diagmicrobio.2021.115422 [Crossref] [ Google Scholar]