Characterisation of genes encoding for extended spectrum β-lactamase in Gram-negative bacteria causing healthcare-associated infections in Mwanza, Tanzania

Healthcare-associated infections (HCAIs) caused by extended spectrum β-lactamase-producing Gram-negative bacteria (ESBL-GNB) increase morbidity and mortality. This cross-sectional study characterised ESBL genes (blaCTX-M, blaTEM and blaSHV) among 30 ceftriaxone-resistant GNB causing HCAIs between January 2022 and July 2022 by multiplex polymerase chain reaction assay at the zonal referral hospital in Mwanza, Tanzania. Twenty-five (83.3%) had at least one ESBL gene, of which 23/25 (92.0%) carried the blaCTX-M gene. Seventy-two percent (18/25) of the GNB-ESBL isolates carried more than one ESBL gene, of which the majority (88.8%; n = 16/25) carried the blaCTX-M and blaTEM genes. Extended spectrum β-lactamase genes, particularly blaCTX-M, are common among ceftriaxone-resistant GNB causing HCAIs. What this study adds This study revealed the distribution of genes (blaCTX-M, blaTEM and blaSHV) coding for ESBL production among ceftriaxone resistant GNB causing HCAIs However, all ESBL producing GNB were susceptible towards ceftriaxone-sulbactam indicating that ceftriaxone-sulbactam may be empirically prescribed for treating patients with HCAIs.


Introduction
Healthcare-associated infections (HCAIs), also referred to as nosocomial infections, are infections acquired by patients while receiving healthcare services from ≥ 48 h after admission to a healthcare facility. 1,2,3 Admission into intensive care units increases the risk of acquiring HCAIs due to (1) chronic diseases which lower body immunity; (2) surgical procedures which interfere with natural body defenses; and (3) medical invasive devices such as urinary catheters, central lines and intubators, which provide bacteria with direct entry into bodily tissues. 4 Escherichia coli, Klebsiella aerogenes, Enterobacter spp., Acinetobacter baumannii and Pseudomonas aeruginosa are the most common Gram-negative bacteria (GNB) known to cause HCAIs. 5,6,7,8 Healthcare-associated infections are associated with significant increased cost of healthcare services, days of hospitalisation and mortality. 9 Healthcare-associated infections caused by multidrugresistant bacteria phenotypes, such as extended spectrum β-lactamase-producing GNB (ESBL-GNB), further exaggerate morbidity and mortality. At the study site in Mwanza, Tanzania, the prevalence of HCAIs in surgical site infections ranges from 10% to 26%. 5,9,10 Staphylococcus aureus accounts for nearly one-third of these, of which about 16% to 19% are methicillin resistant. 5,9 On the other hand, only one study reported 13% of implicating GNB showed ESBL phenotypes. 9 To date, the distribution of ESBL genes among ESBL-GNB phenotypes causing HCAIs is not clearly known. This study unravels the distributions of ESBL genes (bla CTX-M , bla TEM and bla SHV ) among ceftriaxoneresistant GNB causing HCAIs at a zonal referral hospital in Mwanza, Tanzania.

Ethical considerations
This study received ethical approval from the joint Catholic University of Health and Allied Sciences and Bugando Medical Centre Research Ethics and Review Committee. The study approval number is CREC: 2368/2022. All participants voluntarily provided written informed Healthcare-associated infections (HCAIs) caused by extended spectrum β-lactamase-producing Gram-negative bacteria (ESBL-GNB) increase morbidity and mortality. This cross-sectional study characterised ESBL genes (bla CTX-M , bla TEM and bla SHV ) among 30 ceftriaxone-resistant GNB causing HCAIs between January 2022 and July 2022 by multiplex polymerase chain reaction assay at the zonal referral hospital in Mwanza, Tanzania. Twenty-five (83.3%) had at least one ESBL gene, of which 23/25 (92.0%) carried the bla CTX-M gene. Seventy-two percent (18/25) of the GNB-ESBL isolates carried more than one ESBL gene, of which the majority (88.8%; n = 16/25) carried the bla CTX-M and bla TEM genes. Extended spectrum β-lactamase genes, particularly bla CTX-M , are common among ceftriaxone-resistant GNB causing HCAIs. consent before being enrolled in the study. Unique identification numbers were used to ensure confidentiality. Laboratory results were communicated in a timely manner to attending doctors in order to guide rational therapy.

Study design, population, setting and duration
This was a cross-sectional laboratory-based study of ceftriaxone-resistant GNB isolated from different HCAIs between January 2022 and July 2022 (unpublished data) at Bugando Medical Centre -a zonal referral hospital located in Mwanza, Tanzania. The bacterial isolates, which had been archived in 20% glycerol stocks stored in a -40 °C freezer in the Microbiology laboratory as part of a biorepository, were recovered for this study in July 2022. The duration of archive ranged from 1 to 6 months before recovery for molecular characterisation of ESBL genes. Clinical information related to each isolate, namely ward or clinic of origin, sample type, bacterial species name, and susceptibility towards thirdgeneration cephalosporins, notably ceftriaxone, was extracted from the laboratory database. Laboratory procedures were conducted in Microbiology Research Laboratory and Molecular Biology Research Laboratory at the Catholic University of Health and Allied Sciences located at Bugando Medical Centre in Mwanza, Tanzania.

Definition of healthcare-associated infection
In the current study, HCAI was defined as an infection that a patient develops after 48 h of hospital admission, while receiving healthcare for another disease or condition. 11

Recovery of CRO-R-GNB causing healthcare-associated infections and phenotypic detection of ESBL production
Ceftriaxone-resistant GNB causing HCAIs were recovered by sub-culturing on plates of MacConkey agar with salt (MCA; HiMedia, Mumbai, India). Plates were incubated aerobically at 35 °C ± 2 °C for 20 h -24 h followed by phenotypic detection of ESBL production and DNA extraction for multiplex polymerase chain reaction (PCR) assay. The disk combination method (DCM) from the Clinical and Laboratory Standards Institute 12 was used for phenotypic detection of ESBL production among recovered ceftriaxoneresistant GNB.

DNA extraction
From 5 to 10 fresh colonies (≤ 24 h) of ceftriaxone-resistant GNB on plates of MCA were used for DNA extraction. A protocol for DNA extraction from GNB by QIAmp Min DNA extraction kit (QIAGEN, Wuerzburg, Germany) was used according to manufacturer's instructions. DNA samples were stored at −20 °C.

Multiplex PCR assay
A multiplex PCR assay described by Monstein et al. 13 was used for amplification and detection of ESBL genes (bla CTX-M , bla SHV , and bla TEM ). Briefly, 2 µL of each DNA sample was added into a PCR reaction tube containing HotStarTaq DNA polymerase master mix (New England Biolabs; Hitchin, Hertfordshire, United Kingdom) and a set of primers (Table 1), resulting in a final PCR reaction volume of 25 µL. The thermal cycler (T100™, BIO-RAD, Kaki-Bukit, Singapore) was run with the following conditions: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 10 min. Products were detected by using a 1% agarose gel with Trisacetate-EDTA buffer stained with SafeView TM DNA stain (ABM; Richmond, British Colombia, Canada) and visualised under ultraviolet light.

Data management and analysis
Quantitative data were descriptively analysed by using Microsoft Excel (Microsoft Office; Redmond, Washington, United States) and Stata version 15.0 (StataCorp LLP; College Station, Texas, United States; https://www.stata.com/ stata15/).

Results
A total of 30 ceftriaxone-resistant GNB causing HCAIs were recovered during this study period. Most of the recovered bacteria were E. coli 43.3% (n = 13). The majority of ceftriaxoneresistant GNB were isolated from the burn unit (40%; n = 12), and from pus/pus swab samples (56.6%; n = 17). By DCM, all (100%; n = 30) ceftriaxone-resistant GNB had positive ESBL phenotypes. Multiplex PCR assay revealed that about 83.3% (n = 25) had at least one ESBL gene, of which the majority (92.0%; n = 23) harboured the bla CTX-M gene. Out of 25 GNB carrying ESBL genes, 18 (72.0%) carried multiple genes; of these, 88.8% (n = 16) carried bla CTX-M and bla TEM genes ( Table 2 and Figure 1). Five isolates with negative PCR were E. coli (n = 3), isolated from pus/pus swab samples in the burn unit, and Acinetobacter spp. (n = 2), one isolated from a urine sample in the medical ward and the other isolated from a pus/pus swab sample from the neonatal intensive care unit (Table 3).

Discussion
The current study characterised the proportions and distributions of ESBL genes (bla CTX-M , bla TEM , and bla SHV ) among ceftriaxone-resistant GNB which were isolated from different HCAIs between January 2022 and July 2022 at a tertiary zonal referral hospital in Mwanza, Tanzania. The majority of ceftriaxone-resistant GNB were recovered from the burn unit, from patients with burn injuries who were prone to infections because of the breached skin barrier. 14 16,19,20,21 Five confirmed ESBL phenotypes by DCM did not harbour any of the three ESBL genes (bla CTX-M , bla TEM , and bla SHV ) by multiplex PCR assay. This observation is in line with previous studies conducted from the same setting, Mwanza, Tanzania, in 2020 and 2021. 16,17 The isolates may be harbouring other ESBL families which are non-cefotaximase-Munich betalactamase (non-CTX-M), non-temoniera beta-lactamase (non-TEM), and non-sulfhydryl reagent variable betalactamase (non-SHV), such as oxacillinase beta-lactamase, Pseudomonas extended resistant, Vietnam extended-spectrum β-lactamase, Tlahuica Indian-and Guiana-extended spectrum families. 22

Limitations
The small sample size of ceftriaxone-resistant GNB isolates obtained for this study is a weakness but did not affect the interpretation of the results.

Conclusion
Extended spectrum β-lactamase genes, to be specific bla CTX-M , are common among ceftriaxone-resistant GNB causing HCAIs. Therefore, rational management of patients with HCAIs, guided by culture and sensitivity, is warranted.