Molecular characterization and distribution of cephalosporin resistance determinants in Escherichia coli and Klebsiella pneumoniae isolated from patients attending Kampala International University Teaching Hospital in Bushenyi, Western Uganda

ABSTRACT

Cephalosporins are the first-line therapy antibiotics used in the treatment of gram-negative bacterial infections. However, high prevalence of cephalosporins resistance in Klebsiella pneumoniae and Escherichia coli has been reported worldwide. Studies conducted in Uganda reported high incidences of cephalosporin resistance (CR). Successive studies at Mulago National Referral Hospital indicated a decline in the resistance levels pointing to the need for regular antibiotic resistance surveillance. Therefore, this study carried out molecular characterization of CR determinants in E. coli and K. pneumoniae isolated from patients attending Kampala International University Teaching Hospital (KIU-TH). A retrospective study using E. coli and K. pnuemoniae samples previously obtained from surgical wounds and urinary tract infections among patients treated at KIU-TH between September 2016 and August 2018 was conducted. Biochemical assays were used to confirm the identity of the samples. Combined disc and boronic acid assays were used to determine the cephalosporine resistance profile of the isolates. Multiplex PCR amplification was used to characterize the extended spectrum beta-lactmase (ESBL) encoding genes. The study revealed that E. coli (130/81.2%) isolates were more predominant than K. pneumoniae (30/18.8%) among the archived samples. K. pneumoniae showed the highest phenotypic resistance with a mean prevalence of 90.6% but comparable to that of E. coli (89.3%). Of the 160 isolates screened, 105 (65.6%) were ESBL producers. Multiplex PCR revealed that the most predominant ESBL encoding gene was bla_SHV at a prevalence of 42.0%, followed by bla_TEM at 27.3%, bla_CTX-M at 22.4% and bla_CTX-M-15 at 8.4%. The incidence of phenotypic resistance and distribution of ESBL genes were significantly higher in patients of Ishaka division. Our study reports a high prevalence of cephalosporin-resistant E. coli and K. pnuemoniae isolated from patients attending KIU-TH and highlights the need for routine screening of antimicrobial resistance in health-care facilities so as to guide clinicians on the rational prescription of antibiotics.

List of abbreviations: ATCC: American Type Culture Collection; ESBL: extended spectrum beta-lactmases; AmpC: aminopenicillin cephalosporinase; CTX-M: cephotaxime (M-first detected in Munich) hydrolyzing capabilities; CTX-M-U: cephotaxime hydrolyzing capabilities gene Universal primer; SHV: sulfhydryl variables (variant-2); CMY-2: cephamycins (variant-2); TEM: temoneira; ACT-1: AmpC type (variant-1); Bla: Beta lactam; DNA: deoxyribonucleic acid; PCR: Polymerase chain reaction; MNRH: Mulago National Referral Hospital; MRRH: Mbarara Regional Referral Hospital; KRRH: Kabale Regional Referral Hospital (KRRH); KIU-TH: Kampala International University Teaching Hospital; CDL: Central Diagnostic Laboratory; CoVAB: College of Veterinary Medicine, Animal Resources and Biosecurity; CR: cephalosporin resistance

KEYWORDS:

• Escherichia coli
• Klebsiella pneumoniae
• cephalosporin resistance
• extended-spectrum beta lactamases
• ESBL encoding genes

Acknowledgments

We are thankful to the Microbiology Laboratory and Molecular Laboratory, College of Veterinary Medicine Animal Resources and Biosecurity for providing laboratory space.

Availability of data and materials

Supplementary data or any other forms of data have been submitted. Raw data and any other forms data generated by this project can be obtained from the authors on request by e-mail.

Ethics and consent to participate

Ethical Approval No: NrUG-REC-023/201,838 was granted by the Research Ethics Committee for ethical review and approval, Kampala International University-Western Campus. The Research Ethics Committee waived the need for informed consent to use already coded archived samples in this study.

Disclosure statement

The authors declare that they have no competing interests.

Limitations

This study did not look at carbapenem resistance, yet carbapenemase production confers resistance to all beta-lactams. Phenotypic resistance in isolates that neither harboured ESBLs nor AmpCs may be due to the presence of carbapenemase genes. Primers used to detect ESBLs encoding genes in this study targeted most gene variants. Thus, sequencing of the amplicons should be done to distinguish between the several gene variants.

Authors’ contributions

This work was carried out in collaboration between all authors. Eddie Wampande and Kenneth Ssekatawa conceptualized and designed the format for this study. Kenneth Ssekatawa and Herbert Mbyemeire carried out all the laboratory experiments. Kenneth Ssekatawa conducted data analysis. All authors drafted and managed manuscript revisions. All authors read and approved the final manuscript.

Additional information

Funding

The authors declare that this study was funded by the Africa Centre of Excellence in Materials, Product Development & Nanotechnology (MAPRONANO ACE), Makerere University [P151847IDA Credit 5797-UG]. This study was also funded in part by the Swedish International Development Cooperation Agency (Sida), Makerere University under Sida Contribution No: 51180060. The grant was part of the European and Developing Countries Clinical Trials Partnership (EDCTP2) program supported by the European Union.

Notes on contributors

Herbert Mbyemeire

Herbert Mbyemeire is an M.Sc. student and teaching assistant at the Department of Biochemistry, Faculty of Biomedical Sciences, Kampala International University-Western Campus.  Herbert's research interests are in molecular microbiology and antimicrobial resistance.

Kenneth Ssekatawa

Kenneth Ssekatawa is a Research Scientist at MAPRONANO ACE, Makerere University and Lecturer Department of Biochemistry, Faculty of Biomedical Sciences, Kampala International University-Western Campus. Kenneth specializes in molecular microbiology, antimicrobial resistance, phage therapy and nanobiotechnology.

Charles D. Kato

Charles D. Kato is a lecturer at the College of Veterinary Medicine Animal Resources and Biosecurity, Makerere University. Charles' research interests are in biomarker research regarding disease co-infection and host-parasite interactions in infectious and zoonotic diseases. Charles also runs various antimicrobial resistance projects.

Eddie M. Wampande

Eddie M. Wampande is a senior lecturer at the College of Veterinary Medicine Animal Resources and Biosecurity, Makerere University.  Eddie specializes in molecular microbiology and parasitology, as well as antimicrobial resistance.