Role of CTX-M-15 gene in spread of extended-spectrum beta-lactamases among immunocompetent patients in Ghana

Background Patients with faecal carriage of extended-spectrum beta-lactamases (ESBL)-producing Enterobacterales serve as reservoirs and sources of dissemination and infection. Objective This report examined immunocompetent patients for faecal carriage of ESBL-producing Enterobacterales in a district care hospital setting in Ghana. Methods Between March 2019 and May 2020, cross-sectional sampling was performed to enrol patients and conduct questionnaire-structured interviews for factors that predispose patients to ESBL faecal carriage. Faecal samples from study patients were quantified for ESBL-producing Enterobacterales. The ESBL genes were characterised by polymerase chain reaction and sequencing. Results The overall proportion of ESBL faecal carriage was 35.5% (n = 38/107). The blaCTX-M gene, mostly CTX-M-15, was detected in 89.5% (n = 34/38) of the ESBL-producing isolates. The other ESBL types included blaSHV (n = 3) and blaOXA (n = 1). The CTX-M-15-positive isolates, when present in a faecal sample compared to the non-ESBL-CTX-M-15 isolates, constituted the predominant faecal Enterobacterales, with significantly higher colony counts than all other enterobacteria in that sample. In multivariate regression, independent risk factors for faecal carriage of ESBL-producing Enterobacterales were hospitalisation in the past year, infections since admission, use of antibiotics in the past 6 weeks, and admission from another hospital. Conclusion The study found that CTX-M-15-producing isolates were the predominant faecal Enterobacterales, and that further investigations are needed to determine the reasons behind this dominance. What this study adds The CTX-M-15-producing isolates dominance in this study shows the misuse and abuse of antibiotics in an African medical facility and indicates the potential role of immunity in controlling ESBL spread, which is to be investigated further.


Introduction
The presence of extended-spectrum beta-lactamases (ESBL) in Enterobacterales remains the chief resistance mechanism against beta-lactam antibiotics.The ESBL-producing organisms may be widespread in Ghana.About 40% of the Escherichia coli and Klebsiella pneumoniae isolates causing infection in the nation's largest tertiary care facility were found to be ESBL-producing. 1Similar prevalence rates have been reported elsewhere across the country.Infections with ESBLproducing Enterobacterales increase the risk of antibiotic treatment failure, morbidity and mortality, length of hospital stay, and cost of hospitalisation. 2,3ior intestinal colonisation with ESBL-producing isolates has been identified as a significant risk factor for ESBL infections. 4,5These colonised patients remain reservoirs and serve as revolving doors for the spread of resistant pathogens between the community and hospital. 4,5This phenomenon increases the hospital's potential as a repository of multidrug-resistant pathogens, with consequential increases in hospital-acquired infections and its associated economic burden to the patient, hospital and country. 2,3Despite this menace, few studies have reported on the intestinal carriage of ESBLs in Africa, 6,7,8,9,10,11,12,13,14 particularly in Ghana. 15Many advances for reducing the Background: Patients with faecal carriage of extended-spectrum beta-lactamases (ESBL)producing Enterobacterales serve as reservoirs and sources of dissemination and infection.
ESBL menace are proposed in the literature.One such approach involves reducing the effect of patient characteristics that may predispose them to intestinal colonisation with ESBLs. 16,17An important factor that predisposes patients to ESBL intestinal colonisation is the misuse and abuse of antibiotics. 2,18This is more pronounced in immunosuppressed patients where antibiotics are systematically used as prophylaxis to prevent infections.However, there is a knowledge gap about correlations between immunosuppression, immunocompetence, and ESBL intestinal colonisation.Most studies on ESBL colonisation have looked at all patients together, without separating them into groups based on their immune status. 4,6,7,8,9,10,11,12,13,14,15This current study focuses specifically on the occurrence of intestinal colonisation with ESBL-producing Enterobacterales among immunocompetent patients, with particular emphasis on the quantification of ESBL-producing isolates and genotypes of ESBLs.Risk factors for intestinal colonisation with ESBLs were also analysed.
The overarching goal of the current study is to improve our understanding of ESBL faecal colonisation by analysing immunocompetent patients exclusively.The present study is part of a larger explorative study on immunocompetence and ESBL intestinal colonisation.By focusing on this specific group, we hope to provide valuable insights into the dynamics of ESBL colonisation in a subset of patients who have intact immunity.Such knowledge will assist in the development of targeted interventions and strategies to control the spread of ESBL-producing bacteria, improve patient outcomes, and optimise antibiotic usage within this specific patient population.

Ethical considerations
The study was approved by the Ethics and Protocol Review Committee of the School of Biomedical and Allied Health Sciences, University of Ghana (approval number: SBAHS-MD./10551508/AA/5A/2016-2017).Participation in the study was entirely voluntary, and patients were under no obligation to take part.Participating patients provided written informed consent before their enrolment in the study.Interviews were done at the respondent's bedside to ensure privacy.Faecal specimens were assigned arbitrary numbers.Data were aggregated and randomised with unique identification numbers so that no individual could be linked to the study information.

Study design and setting
This was a cross-sectional study to document the presence of intestinal carriage with ESBL-producing Enterobacterales.Faecal specimens were collected, and a questionnaire-based data collection tool (Online Supplementary File 1) was used to conduct interviews at the time of sampling to evaluate patient characteristics that predispose patients to intestinal colonisation with ESBL-producing isolates.The study was conducted from March 2019 through May 2020 at Achimota District Hospital, Accra, Ghana.The hospital is a 100-bed-capacity primary care government facility with no intensive care units.

Study participants and sampling
Before the commencement of the study, appropriate permissions were sought from the hospital authorities.On day 1 of the survey, all patients hospitalised for ≥ 2 days at the Achimota District Hospital were considered potential study participants.An Android app called 'Random Generator' (Apps n Blue, Amman, Jordan) was used to randomly select hospitalised patients to be invited to participate in the study.We assigned each patient a number and entered them into the application.The application generated a random list of numbers, and we approached patients in the order listed.If a patient declined to participate, we moved on to the next patient on the list.On day 2, the patients previously contacted were requested to join the study.Those who agreed to participate were asked to provide informed consent.Participant's medical records were examined with the help of an attending physician to identify patients who were immunocompetent, based on the definitions published by Public Health, England, on immunisation against infectious diseases. 19Patients were considered immunocompetent, in consultations with their attending physician, if they had no major organ or bone marrow transplant, no diagnosis of HIV, no severe combined immunodeficiency, no Wiskott-Aldrich syndrome, and no record of prescription of an immunosuppressive drug or steroid in the month before the sampling date.

Data collection tool
For study participants, a structured data collection instrument (Online Supplementary File 1) was administered to collect demographic, clinical, lifestyle and hospitalisation history information.The tool was adapted from similar studies on ESBL faecal carriage risk factors reported in the literature. 2,16,18e collected data from multiple sources including medical records, direct interactions with the patients, and discussions with their attending doctor.The collected data included patients' demographics (e.g., age, gender, employment status, educational level), which were primarily obtained from medical records.We determined the duration of hospital stays until the survey day through direct patient interactions.Information regarding the type of hospitalisation (hospitalised directly from home or transferred from another health facility) was gathered from discussions with the attending doctors.The number of patients in the participants' ward at the time of recruitment was determined by investigators.Patient lifestyle characteristics, including the use of hand sanitiser at least once in the past three months, travel outside Ghana in the past year, travel outside the home in the past year, and access to pipe-borne water (treated municipal water source) in the household were collected through patient interviews.Information regarding toilet facilities in the household and animal contact in the past three months was also obtained through direct interactions with patients.Data on patient hospitalisation history, including hospitalisation in the past year, use of medication that affects intestinal flora, use of antibiotics in the past six months, and bacterial infections since admission, were extracted from the patients' medical records, discussions with attending doctors, and, where applicable, interactions with patients.

Culture and identification
Patients were given sterile stool containers and instructed to self-collect and submit at least 1 g of stool sample.Participant faecal samples were transported on ice at 0 °C to the microbiology laboratory.At the laboratory, 1 g of each faecal specimen was vortexed in 10 mL of 0.9% sterile saline.Tenfold serial dilutions of each suspension were prepared at 10 -1 to 10 -4 .The serial dilutions were cultured onto Statens Serum Institut enteric age (SSI Diagnostica, Hillerød, Denmark) by mixing 1 mL of each dilution with 24 mL of molten Statens Serum Institut agar (at 52 °C) and incubating aerobically at 35 °C -37 °C for 18 h -24 h.The Statens Serum Institut enteric medium combines selective properties with growth differentiation for direct isolation and rapid diagnosis of Enterobacterales.Quantification was performed by counting growing colonies per enterobacterium morphotype and estimating the number of colony-forming units per gram (CFU/g) of faecal sample.The number of Enterobacterales harboured by each faecal specimen in CFU/g was calculated as the number of colonies per morphotype × 10 1 (for first faecal dilution) × 10 (dilution factor) .Each Enterobacterales morphotype was subcultured onto agar for purity.

Phenotypic determination of extendedspectrum beta-lactamases production
Each colony morphotype of Enterobacterales was examined for ESBL by the combined disk synergy assay.This test was performed using cefpodoxime (30 µg), ceftazidime (30 µg), cefotaxime (30 µg) and cefepime antibiotic discs (30 µg), with and without clavulanate (10 µg), on cation balanced Mueller-Hinton Agar.According to the Clinical and Laboratory Standards Institute interpretative guideline reference, 20 the study isolates that demonstrated clavulanic acid effect defined by an increase in zone diameter greater than 5 mm for at least one test antibiotic were considered ESBL producers.Klebsiella pneumoniae ATCC 700603 (Becton Dickinson, Berkshire, United Kingdom) was used as a positive control for ESBL production.Escherichia coli ATCC 25922 (Becton Dickinson, Berkshire, United Kingdom) was used as a negative control.Only isolates with ESBL producer phenotypes were identified to the species level using the biochemical test kit MINIBACT-E diagnostic (SSI Diagnostica, Hillerød, Denmark), according to the manufacturer's guidelines.

Multiplex polymerase chain reaction for extended-spectrum beta-lactamases genes
Bacterial template DNA for polymerase chain reaction (PCR) was obtained by the boiling suspension method using distilled water as resuspension buffer. 21For the detection of ESBL genes, primers for the sulfhydryl variable active site enzyme (SHV), temoniera enzyme (TEM), oxacillinase 2 and 10 enzymes (OXA-2, OXA-10), and the cefotaxime-Munich (CTX-M) groups 1, 2, and 9 enzymes were used (Online Supplementary File 2).Each PCR mixture was a total of 24.5 µL, constituted as 2 µL template DNA, 12.5 µL of 2 × Multiplex Mastermix (Inqaba Biotec, Pretoria, South Africa), 2.5 µL of 10 × reverse or forward primer each, and 5 µL of DNAse/RNAse free water (Inqaba, Pretoria, South Africa).The PCR conditions 15,22 were initial denaturation at 94 °C for 15 min followed by 30 cycles of amplification for TEM, SHV, OXA-1 and OXA-2 and 27 cycles of amplification for CTX-M group 1, 2, 9 consisting of 30 sec at 94 °C, 1 min at the appropriate annealing temperature for the specific primer (Online Supplementary File 1) and 1 min at 72 °C for primer extension, followed by 10 min at 72 °C for the final extension.The PCR products were analysed on 2% agarose gel (SeaKem ® GTG ® Agarose, Lonza, Basel, Switzerland) alongside a GeneRuler 100 base pairs DNA Ladder Plus marker (Fermentas, Leon-Rot, Germany).

Statistical analysis
Data were analysed using Statistical Package for Social Sciences version 16 (SPSS Inc., Chicago, Illinois, United States, 2007).Comparisons between categorical data were conducted with χ 2 or Fisher's exact tests.Continuous data were compared using a Student's t-test.Point estimates of statistical significance were indicated with two-tailed p < 0.05.Univariate analyses were computed with an odds ratio (OR) with a 95% confidence interval (CI); variables with p < 0.05 were analysed in multivariate logistic regression models to determine independent associated predictor variables(s).The predictive accuracy of the models was evaluated by Hosmer and Lemeshow goodness-of-fit test (http://vassarstats.net/) with p < 0.05 suggesting that the model predicts accurately on average.The area under the Receiver Operating Characteristic Curve > 0.7 was used to analyse the discriminatory capability of ESBL faecal carriage versus their respective controls.

Faecal load of extended-spectrum betalactamases producers
The faecal concentration of ESBL-positive E. coli and K. pneumoniae varied considerably by the type of ESBL gene present (

Univariate comparison of patients' characteristics
Compared to those admitted from home, patients who were admitted from another hospital had a statistically significant

Multivariable logistic regression for independent risk factors
Use of antibiotics in the previous 6 months was the strongest predictor for faecal carriage of ESBL producers (adjusted OR: 3.4; 95% CI: 1.5-10.5;p = 0.0001) (Table 5).Patients with faecal carriage of ESBL producers had used antibiotics 3.4 times more during the preceding six months than patients without faecal carriage of ESBL producers (Table 5).Other independent risk factors were the acquisition of infection(s) since admission (adjusted OR: 3.2; 95% CI: 1.9-7.8;p = 0.002), hospitalisation in the preceding year (adjusted OR: 1.5; 95%

Discussion
In this present study, we examined the occurrence of faecal carriage of ESBL-producing Enterobacterales recovered from immunocompetent patients in a district hospital setting in Ghana.The overall prevalence of faecal carriage by ESBLproducing isolates was 35.5%.The CTX-M genes, mostly bla CTX-M-15 , were the predominant ESBL genotype.When CTX-M-15-producing isolates occurred in a faecal specimen, these isolates were the predominant faecal Enterobacterales.Feglo and Adu-Sarkodie in 2016 (57.8%, n = 234/405), 25 28 It is worth noting that variations in study populations can also influence ESBL carriage rates.For example, the proportion of ESBL faecal carriage reported in our work is lower than that cited for similar populations in comparative reviews 6,17,18 spanning several regions across Africa, including Uganda in 2023 (61%), 29 Ghana in 2022 (53.1%), 30 Kenya in 2022 (44%), 31 Niger in 2022 (92.4%), 32 Malawi in 2021 (47.2%), 33 Central African Republic in 2016 (59%), 10 Morocco in 2014 (42.9%), 14 and Cameroon in 2013 (55.3%). 11In all these studies, the reports were silent on whether the study patients were immunocompetent or immunosuppressed.Indeed, there are limited data on ESBL faecal carriage versus immunity and the paucity of data hinders the ability to compare our findings.Nonetheless, our report provides much-needed data on ESBL carriage rates in immunocompetent patients and serves as a valuable foundation for future studies aiming to compare data and explore the impact of immunity on ESBL carriage rates.This will help us to better understand the factors that contribute to ESBL carriage and to develop strategies for preventing and treating ESBL infections.predominantly CTX-M-15.A possible explanation for the dominance of CTX-M genes could be its ability to localise on large plasmids and co-harbour other resistant genes such as bla AmpC , quinolone-resistance genes or methylase-affecting aminoglycosides. 23The CTX-M-15-borne plasmids in particular have been reported to have a high conjugation frequency and are thus more frequently disseminated to other Enterobacterales species. 24,25The CTX-M genes are transmissible by conjugation with high transfer frequencies of 10 -7 to 10 -2 per donor cell.Perhaps the few TEM, SHV and OXA ESBL types identified in this study point to the fact that CTX-M genes are fast replacing other ESBL types. 26Overall, our report broaches relevant conversations on the possible role of immunity to limiting the spread of ESBL.Will a dominant population of colonising non-ESBL Enterobacterales limit the intestinal occurrence and significance of CTX-M-15?Does the immune status of patients play a role in intestinal colonisation by CTX-M-15 and other ESBL types?Further investigations to ascertain the reasons behind such dominance is warranted.This report is the first of our series to compare ESBL carriage between immunocompetent and immunosuppressed patients.
In multivariable logistics analysis, this study identified antibiotic use in the past 6 months as the highest predictor of ESBL faecal carriage.Several other studies have linked prior antibiotic use within the past 4 or 12 months to an increased possibility of faecal ESBL colonisation. 2,5,15,16,18In our univariate comparisons, patients with ESBL faecal carriage were significantly associated with an acquired infection in the preceding 3 months.The analysis also showed that antibiotic use in the past 3 weeks among patients with ESBL faecal carriage was about four-fold higher compared to that of patients without ESBL faecal carriage.Some studies have even suggested that the more current the antibiotic use, the greater the risk of faecal carriage with an ESBL-positive enterobacteria. 5,17,18These observations are not surprising given that ESBLs and many other antibiotic-resistant mechanisms evolved as a consequence of the misuse and abuse of antibiotics.

Limitations
There are some potential limitations of this study that should be discussed briefly.Bacterial clonal relatedness was not investigated in this work.Such investigations would have enabled the study to compare various ESBL faecal enterobacteria to determine the extent to which these isolates are being disseminated.Another noteworthy limitation is the rather few study patients.A more large-scale survey is much more likely to be with little bias for high-at-risk ESBL faecal carriers.

Conclusion
The findings of this study suggest that faecal carriage of ESBL-producing Enterobacterales is a significant public health concern in Ghana.The high prevalence of ESBLproducing bacteria, the association with antibiotic use, and the identification of CTX-M-15 genes as a potential driver of multidrug-resistant bacteria dissemination in immunocompetent patients are all concerning trends.These findings highlight the need for increased surveillance of ESBL-producing bacteria in Ghana and other parts of Africa.Further research is needed to explore the relationship between ESBL faecal carriage and immunity, understand the dominance of CTX-M genes, and address the consequences of antibiotic misuse.More research is also needed to better understand the factors that contribute to ESBL faecal carriage and to develop effective interventions to prevent the spread of these bacteria.

TABLE 2 :
Specific types of extended-spectrum beta-lactamases gene sequences in Escherichia coli and Klebsiella pneumoniae in Accra, Ghana, March 2019 to May 2020.

TABLE 3 :
Comparison of the faecal concentration (colony-forming units/g) of extended-spectrum beta-lactamase-positive and all extended-spectrum beta-lactamasenegative Enterobacterales bacteria in faecal samples of 38 extended-spectrum beta-lactamase faecal carriers in Accra, Ghana, March 2019 to May 2020.

TABLE 4 :
Univariate comparison of the risk factors among patients with and without ESBL-positive faecal carriage in Accra, Ghana, March 2019 to May 2020.

TABLE 5 :
Independent risk factors for faecal carriage with extended-spectrum beta-lactamase-producing Enterobacterales among study participants using multivariate logistic regression analysis in Accra, Ghana, March 2019 to May 2020.

TABLE 4 (
continues ...): Univariate comparison of the risk factors among patients with and without ESBL-positive faecal carriage in Accra, Ghana, March 2019 to May 2020.
This research has been supported by the University of Ghana -Carnegie Building a New Generation of Academics in Africa Project with funding from the Carnegie Corporation of New York (Grant number: UG-BA/PD-016/2017-2018, principal investigator N.O.-N).