Antibiotic Resistance Profiles of Diarrhoeagenic Enterobacterales in Bioko Island, Equatorial Guinea

Abstract

Acute diarrhoeal disease caused by antibiotic-resistant diarrhoeagenic bacteria is a significant global public health issue, particularly in low- and middle-income countries. This study provides the first molecular characterisation of antimicrobial resistance profiles, including the detection of CTX-M-15 and CTX-M-55 extended-spectrum beta-lactamases (ESBLs), among diarrhoeagenic Enterobacterales in Bioko Island, Equatorial Guinea, offering novel epidemiological insights into an understudied region. This study investigated the antibiotic resistance profiles of pathogenic bacteria isolated from diarrhoeal samples on Bioko Island. A total of 153 clinical isolates were collected between 1 February and 30 May 2014, and antimicrobial susceptibility testing was performed at Loeri Comba Polyclinic (Malabo) using the Kirby–Bauer method. The molecular characterisation of β-lactamase-associated genes was performed on different isolates of diarrhoeagenic pathotypes—144 Escherichia coli, 7 Salmonella enterica, and 2 Shigella flexneri—at the National Centre for Microbiology (Majadahonda, Spain). High resistance rates were detected against ampicillin (98%), tetracycline (93.5%), sulfonamides (94.8%), sulfamethoxazole–trimethoprim (88.2%), and cefotaxime (78.8%), while moderate rates of resistance were noted for ciprofloxacin (26.7%), and all isolates remained susceptible to imipenem. Of the isolates, 107 (69.9%) produced either single or multiple β-lactamases. Among these, 73 (68.2%) harbored classical β-lactamases, specifically TEM and OXA-1 types, representing 47.7% of the total sample. Additionally, 34 (31.8%) of the isolates were identified as producers of extended-spectrum β-lactamases (ESBLs), specifically CTX-M enzymes. Sequencing identified CTX-M-15 and CTX-M-55 variants. The predominant ESBL-producing bacteria were enteroaggregative Escherichia coli (56.2%), followed by enteropathogenic and enterotoxigenic E. coli. These findings confirm the circulation of multidrug-resistant diarrhoeagenic Enterobacterales in Equatorial Guinea, raising concerns about limited treatment options due to widespread resistance to multiple antibiotic classes, including third-generation cephalosporins and quinolones. The most important conclusion drawn from this study is that a high percentage of diarrhoeagenic bacteria have an antibiotic resistance and multi-resistance profile, especially to beta-lactams and other groups of antibiotics such as tetracyclines and sulphonamides. There is also a moderate prevalence of isolates carrying ESBLs on Bioko Island, Equatorial Guinea, which could indicate the inappropriate use of antimicrobials.

1. Introduction

The WHO reported a few years ago that by 2050, infectious diseases caused by multidrug-resistant bacteria will account for more deaths than cardiovascular disease and cancer combined [1]. For years, it has been urging countries to work together to find viable solutions to combat these pathogens, as they pose one of the greatest threats to public health, food security, and social and economic development in the world.

Bacterial isolates of multidrug-resistant strains are increasingly common in lower-middle-income countries [2,3]. Of the few investigations in Africa, studies in Tanzania and Kenya reported a worrying increase in pathogenic Enterobacteriaceae resistant to broad-spectrum antibiotics such as third-generation cephalosporins, aminoglycosides, and quinolones [3,4]. Other studies in Burkina Faso and Nigeria [5,6] not only reported high resistance to multiple antibiotics but also raised concerns about the increasing use of broad-spectrum antibiotics to treat infections with ESBL-producing bacteria, with the ability to generate strains with a much higher resistance profile to existing antimicrobials, making treatment more difficult when needed [7].

Some African studies [6,8] also reported an alarming increase in infections with ESBL-carrying bacteria, mainly due to their high and multiple antibiotic resistance capacity and how easily they spread in communities.

In Equatorial Guinea, as in many African countries, there is not a sufficient documented history of research on bacteria that cause multidrug-resistant diarrhoea, so the results of this study are of great interest and could be used to evaluate and compare the epidemiological situation on Bioko Island in recent years. Equatorial Guinea shares risk factors with many African countries that favour the increase and dissemination of antibiotic multidrug-resistant strains, such as the indiscriminate use of these drugs, the availability of these drugs without medical prescription in public and private establishments, non-compliance with treatment guidelines, or poor monitoring to assess the evolution of infections caused by resistant strains, among others [8,9]. Various studies conducted in African countries—including Tanzania, Burkina Faso, Nigeria, Cameroon, Gabon, Ethiopia, and Chad—have documented a growing prevalence of multidrug-resistant enteric bacteria, especially Escherichia coli, Shigella, and Salmonella isolated from diarrhoeal samples. Recent research has identified high levels of resistance to beta-lactams, sulphonamides, tetracyclines, and quinolones, as well as the circulation of extended-spectrum beta-lactamase (ESBL) genes, such as bla_CTX-M, bla_TEM, and bla_SHV. These findings consistently indicate that both hospitalised patients and apparently healthy children can carry multidrug-resistant strains, representing a silent community reservoir that exacerbates the problem. The uncontrolled availability of antibiotics, lack of surveillance programmes, and limited therapeutic options in these contexts pose a serious public health challenge, highlighting the urgent need to implement coordinated measures to contain the spread of resistant strains in Africa.

This study was conducted to understand the antibiotic resistance profiles of diarrhoeagenic pathogenic bacteria on Bioko Island, which will serve as a basis for future research on antibiotic-resistant acute diarrhoeal disease in Equatorial Guinea.

2. Materials and Methods

2.1. Study of Antimicrobial Resistance Profiles in Diarrhoeagenic Enterobacterales

Study Area and Population

An antimicrobial susceptibility study was conducted on Bioko Island between 1 February 2014 and 30 May 2014. Bioko Island is part of the Republic of Equatorial Guinea, which also includes Rio Muni on the mainland and the island of Annobón. Bioko Island has 334,463 inhabitants and an area of approximately 2017 km² (779 sq mi). It is 72 km long and is divided into four districts (Malabo, Luba, Riaba, and Baney). Most of the population lives in the northern part of the island, in the district of Malabo, where the capital of Equatorial Guinea is located. The island is divided into two provinces: Bioko Norte and Bioko Sur. The study analysed a total of 153 samples from patients with diarrhoeal disorders associated with diarrhoeagenic bacteria from the two reference hospitals on the island, the Loeri Comba Polyclinic and the Malabo Regional Hospital, which provide direct medical care to the 132,440 inhabitants of Malabo’s urban and peri-urban populations. Patients of all ages presented with acute diarrhoea (lasting less than 14 days). Inclusion criteria: confirmed presence of infectious diarrhoea based on clinical evaluation and/or laboratory tests; no antibiotic treatment received within 72 h prior to stool sample collection; resident of Bioko Island for at least 6 months prior to the illness; signed informed consent obtained (from the patient or legal guardian for minors); and stool samples collected at the reference hospitals (Loeri Comba Polyclinic and Malabo Regional Hospital).

In line with the Loeri Comba Polyclinic Laboratory protocol, acute diarrhoeal disease (ADD)-associated bacteria were characterised by biochemical methods (18R, Liofilchem, Italy; API, Biomerieux, France) and confirmed at the National Centre of Microbiology by PCR. The validation of quality assurance (QA) and quality control (QC) for the molecular detection of antimicrobial resistance genes was carried out at the Reference Laboratory for Food and Waterborne Diseases: Enterobacteria Unit, National Microbiology Centre, Instituto de Salud Carlos III (Madrid, Spain). The laboratory implemented rigorous internal and external quality controls in accordance with ISO 15189 standards (https://www.iso.org/standard/76677.html, accessed on 15 April 2020) to ensure the accuracy and reproducibility of the PCR-based identification of bla_TEM, bla_CTX-M, bla_SHV, and bla_OXA-1 genes. Validation included the use of certified reference strains with known resistance profiles as positive and negative controls in each PCR run, as well as replicate testing to confirm inter-assay consistency. Additionally, the sequencing of representative amplicons was performed to verify the specificity of the PCR assays. The laboratory participates regularly in international proficiency testing schemes to maintain high-quality standards in molecular diagnostics (http://www.scielo.org.mx/pdf/spm/v44n5/14036.pdf, accessed on 15 April 2020). Antibiotic susceptibility testing was conducted using the Kirby–Bauer method, and the cut-off points recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were followed. In cases without defined cut-off points, those indicated in the CLSI guidelines were followed [10]. The study used the antibiotics recommended by the ECDC for antimicrobial susceptibility surveillance. If resistance or decreased susceptibility to second-generation cephalosporins was detected, double synergy testing confirmed the presence of β-lactamases by the diffusion of amoxicillin–clavulanic acid (20/10 μg), cefotaxime (30 μg), and ceftazidime (30 μg) discs; presumptive ESBL isolates were those in which a synergistic effect between the AMC inhibitor and the CTX or CAZ discs was observed by broadening the inhibition halo of cefotaxime or ceftazidime, in the area close to the amoxicillin–clavulanic acid disc (synergy), or the presence of a ‘phantom zone’. Genetic resistance determinants were identified by PCR through amplifying the TEM, CTX-M, and SHV families and subsequent sequencing [7]. In the case of ampicillin–clavulanic acid and cefoxitin resistance, OXA-1 and CMY were also tested [2].

3. Results

Of the 153 samples analysed, 63.4% (97/153) were obtained from the Loeri Comba Polyclinic and 36.6% (56/153) from the Regional Hospital. The majority of isolates (94.1%, n = 144) were enterobacterales belonging to different pathotypes of Escherichia coli. Among these, 56.2% (n = 86) were identified as enteroaggregative E. coli (EAEC), 20.9% (n = 32) as enterotoxigenic E. coli (ETEC), 16.3% (n = 25) as enteropathogenic E. coli (EPEC), and 0.7% (n = 1) as enteroinvasive E. coli (EIEC). The remaining isolates included Salmonella enterica (4.6%, n = 7) and Shigella flexneri (1.3%, n = 2).

3.1. Antibiotic Resistance Profiles

The 153 isolates showed high rates of resistance to β-lactams, especially penicillins and first- and second-generation cephalosporins, with 98% of isolates resistant to at least one of these and 40.1% resistant to all of them. All isolates were susceptible to imipenem. The highest rates of resistance were observed for ampicillin (98.03%), cefotaxime (78.8%), amoxicillin–clavulanic acid (71.24%), cephalothin (56.9%), ceftazidime (13.07%), and cefoxitin (9.1%). This study also found high rates of resistance to antibiotics from other classes, such as tetracycline (93%), cotrimoxazole [sulfamethoxazole–trimethoprim] (88.2%), sulphonamides [sulphamethazine, sulphathiazole, sulphadiazine] (94.8%), and streptomycin (73.2%). Moderate rates of resistance were noted for ciprofloxacin (26.7%), nalidixic acid (42.4%), and chloramphenicol (36.6%). The lowest resistance rates were found for gentamicin (15%) and kanamycin (13.7%).

Of the 153 isolates tested, 90 (58.8%) presented resistance to three or more antibiotic families, qualifying as multidrug-resistant. This resistance was particularly notable against the most used antibiotics in Equatorial Guinea. Among these multidrug-resistant isolates, the highest resistances rates were observed for β-lactams, including ampicillin (100%) and amoxicillin–clavulanic acid (93.3%), as well as tetracycline (98.9%) and cotrimoxazole (96.7%).

Table 1. General resistance rates.

Antibiotic	Resistance Rate (%)
Ampicillin	98.03
Cefotaxime	78.8
Amoxicillin–clavulanic acid	71.24
Cephalothin	56.9
Ceftazidime	13.07
Cefoxitin	9.1
Tetracycline	93.0
Cotrimoxazole (SXT)	88.2
Sulphonamides	94.8
Streptomycin	73.2
Ciprofloxacin	26.7
Nalidixic acid	42.4
Chloramphenicol	36.6
Gentamicin	15.0
Kanamycin	13.7
Imipenem	0.0

,

Table 2. Resistance by isolate type.

Isolate Type	Multidrug Resistance (%)
EAEC	70.0
ETEC	60.0
EPEC	50.0
EIEC	0.0
Salmonella enterica	28.6
Shigella flexneri	50.0

and

Table 3. Resistance genes by bacterial pathotype.

Pathotype	TEM	CTX-M	OXA-1	Total Isolates
EAEC	19	20	1	40
EPEC	12	10	0	22
ETEC	17	0	2	19
EIEC	0	0	0	0
Salmonella enterica	1	0	1	2
Shigella flexneri	0	1	0	1

present a breakdown of the antibiotic resistance profiles, while

Table 4. Characterisation of beta-lactamase genes in diarrhoeagenic bacterial isolates.

Pathotype/Species	TEM	CTX-M	OXA-1	Multiple Genes
EAEC	19	20	1	20
EPEC	12	10	0	3
ETEC	17	0	2	0
EIEC	0	0	0	0
Salmonella enterica	1	0	1	0
Shigella flexneri	0	1	0	0

provides a characterization of the β-lactamase genes in the diarrhoeagenic bacterial isolates.

3.2. Characterisation of Beta-Lactamase Genes in Diarrhoeagenic Bacterial Isolates

To characterise the genes responsible for the production of enzymes associated with β-lactam resistance, PCR was performed using universal primers targeting the genes bla_TEM, bla_CTX-M, bla_SHV, and bla_OXA-1. Out of a total of 153 isolates, the study identified the presence of beta-lactamases in 107 (69.9%) of them, while 46 (30.1%) did not exhibit these enzymes. Among the beta-lactamase carriers, 73 (68.2%) showed the presence of classical beta-lactamases, specifically TEM and OXA-1, which represent 47.7% of the total sample. On the other hand, 34 (31.8%) of the isolates, corresponding to 22.2% of the total, were identified as carriers of extended-spectrum beta-lactamases (ESBLs), specifically CTX-M enzymes.

Of the 153 isolates, 103 (96.2%) were DEC, 3 (2.8%) were Salmonella enterica, and 1 (0.9%) was Shigella flexneri. A total of 84 isolates (78.5%) carried a single β-lactamase gene, while 23 (21.5%) harboured multiple β-lactamase genes. Among the 84 isolates with a single class of β-lactamase, the most common types were TEM (n = 51) followed by CTX-M (n = 31) and OXA-1 (n = 2). The distribution of these β-lactamases across carrier pathogens was as follows: 40 EAEC isolates (including 19 with TEM β-lactamase, 20 with ESBL CTX-M, and 1 with OXA-1); 22 EPEC isolates (comprising 12 with TEM β-lactamase and 10 with CTX-M type); and 19 ETEC (with 17 carrying TEM β-lactamases and 2 with OXA-1). No β-lactamase genes were identified in the EIEC pathotype. In Salmonella enterica, two isolates of the S. typhimurium 4,5,12:i:-monophasic variant exhibited β-lactam resistance, with one carrying a TEM β-lactamase and the other an OXA-1 β-lactamase. Lastly, we identified CTX-M in a single isolate of Shigella flexneri.

The 23 isolates identified as carrying more than one type of β-lactamase gene were associated with DEC pathotypes. Of the 20 EAEC isolates, 19 carried both TEM and OXA-1 genes (n = 19), and one carried CTX-M and OXA-1. Additionally, three EPEC isolates harboured multiple β-lactamase genes, with the following combinations identified: TEM and CTX-M, TEM and OXA-1, and CTX-M and OXA-1. We did not identify any isolates with more than one type of β-lactamase gene in Salmonella enterica and Shigella flexneri. Among β-lactamase-positive isolates, 34 (31.8%) were identified as ESBL producers, which represents 22.2% of the total 153 isolates. Table 4 shows the characterisation of beta-lactamase genes in diarrhoeagenic bacterial isolates.

3.3. Sequencing of β-Lactamase Genes from Diarrhoeagenic Bacterial Isolates

The sequencing revealed that all the ampicillin-resistant isolates carried the TEM-1 gene. The phenotypic double synergy test was positive in 34 isolates that had been identified as producers of CTX-M-type β-lactamases. Sequencing revealed that of the 21 EAEC isolates positive for CTX-M, 18 contained CTX-M-15 and only 3 had CTX-M-55. Meanwhile, the 12 EPEC and the Shigella flexneri isolate exhibited CTX-M-15.

The antimicrobial resistance of isolates with the ESBL genotype comprised ampicillin (98.03%), streptomycin (97.3%), cephalothin (100%), nalidixic acid (100%), cefotaxime (100%), tetracycline (100%), amoxicillin–clavulanic acid (97.3%), sulfamethoxazole–trimethoprim (100%), ciprofloxacin (88.9%), kanamycin (42%), chloramphenicol (50%), and sulfonamides (100%). All ESBL-carrying isolates had a multi-resistance profile.

4. Discussion

Since the discovery of penicillin, bacteria have evolved and developed different mechanisms to cope with the antibiotics used to treat infections. β-lactam antibiotics are the most widely used antibiotics in clinical practice, and there are already a multitude of resistant strains. In low- and middle-income countries, sulfamethoxazole–trimethoprim, ampicillin, and tetracycline are widely used to treat diarrhoea and other bacterial infections, which has increased resistant strains [11].

Our study detected high resistance of diarrhoeagenic bacteria to β-lactam antibiotics and other commonly used antibiotic families on Bioko Island. These results are similar to those obtained by Mafo et al. [11] and Bay et al. [12] in other geographical areas, who reported an increase in the frequency of penicillin resistance, with the difference being that the latter reported low resistance of E. coli to amoxicillin–clavulanic acid. In this study, we would like to highlight the high prevalence of multidrug-resistant isolates, with a resistance profile similar to those reported in other studies in neighbouring countries [6,11], which is of great concern in the area. It should be noted in this study that although there are no previous studies on antibiotic resistance in Enterobacteriaceae in Equatorial Guinea, there is evidence in the archives of the PLC (Malabo) and publications in other countries in the region, such as Nigeria [6] and Gabon [13], that ampicillin, sulfamethoxazole–trimethoprim, and amoxicillin–clavulanic acid have been the most widely used and abused antibiotics in the region, probably because they are cheaper and available without a prescription at any pharmacy [11,14].

The results obtained concerning multi-resistance in this study reflect a situation of great concern in the therapeutic use of antibiotics, mainly ampicillin, cotrimoxazole, and amoxicillin–clavulanic acid. In cases where their use is necessary in treating infections caused by enterobacterales in Equatorial Guinea, the antibiotics mentioned above would not be sufficiently effective, which coincides with the findings of studies carried out by other authors [3,15]. Notably, while the overall resistance rates for amoxicillin–clavulanic acid and cotrimoxazole were 71.24% and 88.2%, respectively, these rates were significantly higher within the multidrug-resistant subset. This suggests that multidrug-resistant isolates exhibit increased resistance to commonly used antibiotics in Equatorial Guinea, further limiting treatment options.

Regarding first- and second-generation cephalosporins, we found significant variation in resistance to cefoxitin and cephalothin. Our isolates showed low resistance to third- and fourth-generation cephalosporins, which is in agreement with data from M. Shah et al. [16], who also reported low resistance of DEC to these drugs, although at slightly lower rates than those obtained by us. Konate et al. [5] in Burkina Faso in 2017 reported high resistance to third-generation cephalosporins in DEC. In comparing our data with previously cited studies, there are differences in DEC resistance to third-generation cephalosporins and fourth-generation cephalosporins depending on the geographical area. In our case, 3G cephalosporins or ciprofloxacin could be used for the treatment of gastroenteritis when necessary on Bioko Island, given the low resistance rates observed among the study isolates, these antibiotics may be considered beneficial for use in order to help prevent the further development of antimicrobial resistance. The isolates were generally moderately resistant to other antibiotics, such as chloramphenicol and quinolones, and showed low resistance to aminoglycosides such as gentamicin, which is among the antibiotics also used in cases of bacterial diarrhoea in many African countries, such as Burkina Faso or Cameroon [5,11]. All the isolates studied showed 100% sensitivity to imipenem, probably because its use is very recent in the country’s therapeutics, being limited for the time being to hospital use (unpublished data from the Ministry of Health). Similar resistance patterns among the different pathotypes of diarrhoeagenic bacteria could suggest a high rate of horizontal transfer of resistance genes among these bacteria and highlight the need to establish adequate control and prevention measures [17,18]. Therefore, the identification of multidrug-resistant bacteria in our study underscores the selective pressure resulting from the widespread and inappropriate use of β-lactam antibiotics, which are commonly employed to treat bacterial infections in Equatorial Guinea. This finding emphasises the urgent need for national measures to regulate antibiotic usage, alongside the implementation of a countrywide surveillance system for antibiotic-resistant bacteria, to inform and establish effective empirical treatment guidelines. This study found high resistance to β-lactams and multi-resistance to different families of antibiotics in the group of diarrhoeagenic bacteria, and EAEC was the pathotype where the most resistance and ESBLs were detected, probably due to the indiscriminate use of these drugs for years in Equatorial Guinea and other African countries, as they are cheaper and easily accessible because they do not require a medical prescription (unpublished data, [8]). In our study, we found that the E. coli and Shigella flexneri pathotypes were capable of producing CTX-M ESBLs as well as isolates carrying combinations of resistance genes to different β-lactams, including CTX-M+TEM-1 and CTX-M+OXA-1; this could explain the high levels of resistance to β-lactam antibiotics found in this study, because, as some authors have noted [9,11], the existence of specific associations of resistance genes in strains, especially enterobacterales, increases their resistance to extended-spectrum antibiotics. Other studies suggest that ESBL-encoding genes are often associated with resistance genes from other antibiotic groups, giving rise to multi-resistance, especially to aminoglycosides and quinolones [19]. In our case, the isolates did not show high resistance to the latter but did resist tetracyclines and sulphonamides. Resistance to β-lactams has serious implications for the treatment of bacterial diarrhoeas in our context. β-lactams, especially cephalosporins, are often the treatment of choice for severe infections. Resistance to these drugs restricts therapeutic options, forcing doctors to resort to second-line or last-generation antibiotics, which may be less accessible or more costly. When bacteria are resistant to broad-spectrum β-lactams, doctors must use antibiotics like carbapenems, which are expensive and have limited availability in African hospitals. This raises healthcare costs and can compromise the availability of these antibiotics for other patients. Additionally, resistance reduces treatment effectiveness, which can increase the duration and severity of diarrhoeal infections. This is particularly concerning in vulnerable populations, such as children under five, who are especially susceptible to the effects of severe diarrhoea. Finally, antibiotic resistance in diarrhoea-causing bacteria can lead to higher morbidity and mortality due to reduced treatment efficacy and an increase in secondary infections. Severe infectious diarrhoea that is not adequately treated can cause severe dehydration and additional complications, raising the mortality rate in communities with limited access to healthcare services. This study coincides with several previous studies in other countries, which raise the same concern about the increase in multidrug-resistant bacteria and ESBL carriers [18,19], a severe problem for health systems, as it limits treatment options [7]. In our study, 58.8% of the isolates were multidrug-resistant, and 11.8% of these harbored ESBL genes, similar to results reported in other studies [6]. The ESBL-bearing isolates identified showed resistance to all penicillins and cephalosporins and to β-lactam combinations (AMCs), as well as to other antibiotic families; however, they were sensitive, in most cases, to cephamycins and fully sensitive to carbapenems. In these cases, carbapenem therapy, which is difficult to access in various African countries, could be applied if necessary.

Although ESBL-producing bacteria are recognised as a severe problem in Africa, reporting is low due to a lack of resources [5]. This study reveals a high prevalence of multidrug-resistant diarrhoeal bacteria, with many carrying ESBLs, particularly TEM-1 β-lactamases, which hydrolyse penicillins and cephalosporins. Though not classified as ESBLs, these β-lactamases hold clinical and epidemiological significance due to their potential mutations that enhance activity against extended-spectrum β-lactams [12,15]. Globally, bacterial resistance varies by region [11,16]. While CTX-M-type ESBL is increasingly dominant worldwide, TEM-1 was the most frequent type in our study. However, studies from Europe, Latin America, and Canada indicate CTX-M as the most prevalent and rapidly spreading ESBL type. In our samples, ESBLs were primarily found in DEC pathotypes and a Shigella isolate, with CTX-M being the most common. This type, particularly CTX-M-15, has successfully spread globally, especially via E. coli [17], and is prevalent in Europe, South America, and parts of Africa, including Nigeria and Cameroon [6,11,18]. An increase in Enterobacterales carrying CTX-M ESBLs has been reported worldwide [17]. Although our study found a high prevalence, it remains lower than in other regional studies [6,11]. We sequenced two CTX-M variants, with CTX-M-15 being the most common—consistent with previous reports [14]—and CTX-M-55 appearing less frequently but being previously identified in South Asia [18]. The widespread presence of CTX-M-15 in Equatorial Guinea mirrors trends in neighbouring countries [6,13], highlighting its significant dissemination potential [17] and rising concerns for regional public health.

5. Limitations of the Study

The limited sample size and the restriction of the study to Bioko Island restrict our ability to fully assess the extent of ESBLs within the Guinean population. Therefore, expanding the sample in future research would be crucial for a more comprehensive understanding of the epidemiological distribution of ESBL-carrying strains in Equatorial Guinea. The 14 isolates with a cefoxitin-resistant ESBL phenotype could indicate the presence of a gene coding for AmpC-type β-lactamases other than CMY, which was not confirmed as it was not a target in the present study. The lack of data on prior antibiotic use by patients is significant, as such use may have selected for the multidrug-resistant strains identified if it preceded the onset of diarrhoea. Another limitation is the lack of epidemiological data to distinguish between isolated and related cases (outbreak context). Thus, in cases of related samples, a particular strain/resistance mechanism would be over-represented.

6. Conclusions

The most important conclusion drawn from this study is that a high percentage of diarrhoeagenic bacteria have an antibiotic resistance and multi-resistance profile, especially to β-lactams and other groups of antibiotics such as tetracyclines and sulphonamides. There is also a moderate prevalence of isolates carrying ESBLs on Bioko Island, Equatorial Guinea, which could indicate the inappropriate use of antimicrobials. The presence of ESBLs in diarrhoeagenic bacteria in this study limits the treatment options for necessary cases, so it is essential to implement measures to prevent these infections, carry out more studies on the prevalence of ESBLs and antimicrobial resistance, restrict the use of antimicrobial agents with measures aimed at preventing the sale of antibiotics without a doctor’s prescription and public awareness campaigns. The timeframe of this study may prompt questions about its current relevance. However, given the lack of a nationwide surveillance system for antibiotic-resistant bacteria in Equatorial Guinea, it is likely that this issue persists. We recommend conducting new investigations to evaluate the current spread of ESBLs among circulating bacterial strains in the country, utilising advanced molecular techniques to enhance our understanding in this field.