ESBL and carbapenemase-producing Enterobacteriaceae in infectious pleural effusions: current epidemiology at Hôpital du Mali

Introduction

Infectious pleural effusion (IPE) is a severe clinical issue. Often secondary to a pre-evolving pulmonary infection, the condition presents with an increasing incidence worldwide (1,2). Care of IPE involves pleural drainage in most of the cases and antibiotic therapy; however, treatment proves inadequate in substantial cases, resulting in mortality rate ranging from 10.7% to 22% (3,4,5). Few studies have evaluated IPE management, status, and microbiology in Mali. A recent study by Tapia et al in 2021 reported 13.3% mortality rate (6). Among causal factors predictive of treatment failure is the alarming emergence of antimicrobial resistance (AMR).

AMR has emerged as a significant global health concern, undermining the effectiveness of antibiotics and exacerbating the burden of infectious diseases. Major contributors to AMR include extended-spectrum β-lactamases (ESBLs) and carbapenemases produced by Enterobacteriaceae (7,8). Both enzymes are β-lactamases with proven ability to degrade β-lactam antibiotics. The ESBLs exhibit hydrolytic activity against penicillins and all cephalosporins, yet are suppressed by β-lactam inhibitors (9). Mutation-wise, ESBLs encoding genes can be grouped into several variants: bla_TEM, bla_SHV, bla_IRT, etc (10). On the other hand, another important enzyme is carbapenemase, which degrade carbapenem antibiotics and include gene variants like bla_KPC, bla_IMP, bla_OXA-1, etc (9). Thus, their inclusion in World Health Organization list as top priority pathogens underscores their potential to pose significant challenge in clinical settings (11). Data from recent studies found an association between extended-spectrum β-lactamases and carbapenemases producing Enterobacteriaceae (ESBL-E/CPE) infections and risk of mortality (12,13). Moreover, prior to antibiotherapy, comorbidities as well as persistent colonization were found to be strong predictors of infection and subsequent treatment failure (14,15). Yet to date, detailed reports on the magnitude of ESBL-E/CPE in West Africa are scarce (16). Although there are data on phenotypic and genotypic distribution of AMR in pathogens responsible for various clinical infections in Mali, there are still unanswered questions related to the genotypic distribution of these AMR genes in IPEs (17,18,19). Understanding the extent of resistance and identifying the underlying resistance mechanisms is crucial for designing effective therapeutic strategies and optimizing patient care in this region.

This study has mainly focused on the epidemiology of bla_OXA-48, bla_NDM-1, bla_KPC genes responsible for the induction of carbapenem resistance; bla_TEM, bla_SHV genes that are responsible for ESBL production, as well as genes associated with bacterial adhesins (bfp, eae, ipah, eagg) and toxin genes (slt1, slt2, lt, sta). The primary objective of this study was to isolate, identify, and analyze the diversity of enterobacterial species found in pleural fluid samples from patients with IPE. A better knowledge of IPE in Mali could facilitate the management of the condition and thus reduce the level of mortality.

Material and methods

Molecular analysis

DNA was extracted according to the method described previously (22). Briefly, pure colonies were suspended in 200 μL of Tris-ethylenediamine tetraacetic acid (EDTA) solution, heated at 100°C for 10 minutes, and then immediately placed at −20°C for 5 to 10 minutes. After centrifugation at 12,000 rpm for 10 minutes, the obtained supernatant was used as DNA template. Quality control of the extraction was carried out using Thermo Scientific NanoDrop One/One^C instrument at the molecular biology unit of the University Centre for Clinical Research (UCRC) in Bamako.

Using conventional polymerase chain reaction (PCR), eight virulence factor genes from Escherichia coli, namely bfp, eae, eagg, ipah, slt1, slt2, lt and sta, as well as ESBL coding genes, namely bla_TEM and bla_SHV, were characterized. In addition, the following genes were screened for their role in conferring antibiotic resistance: the catA1 gene responsible for encoding chloramphenicol acetyltransferase, mutations on genes encoding for topoisomerase IV, and DNA gyrase protective proteins targeted by quinolones (qnrA, qnrB, qnrS), as well as class 1 (int1), class 2 (int2), and class 3 (int3) integrons, which carry resistance genes for multiple antibiotics. We screened bacterial isolates for the presence of carbapenemase genes, namely: bla_KPC, bla_NDM-1, and bla_OXA-48.

PCR was performed using ABI 9700 thermocycler (Applied Biosystems, USA) with the following cycling parameters: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at variable temperature for each primer set for 39 seconds, and extension at 72°C for 60 seconds (Tab. II). Table II provides essential information about selected genes, including their nucleotide sequences, hybridization temperatures, amplicon sizes. The final extension step was carried out at 72°C for 5 minutes. Each reaction was carried out in a 25 μL mixture prepared as described previously, with modifications (22). In all reactions, a negative control (water) was included alongside a positive control consisting of reference isolates (E2348-69, M90T, EDL 933, EDL 1493, R3, R4, R5, R6, and R7).

The sequence of primers used for amplification and the expected amplicon size are detailed in Table II. PCR products were visualized by transillumination after migration in 1.5% Tris base, acetic acid and EDTA (TAE) buffer.

Results

Discussion

The results reported in this study provide valuable insights into the bacteriology of PIs in Mali. By addressing resistance rates toward various antibiotics, and the frequency of ESBL, carbapenemases, and MDR bacteria, this study is the first to comprehensively analyze these factors and lay the groundwork for future clinical studies to determine whether improved bacterial diagnosis and antibiotic selection can positively impact the outcomes of PIs. According to the British Thoracic Society (BTS) Guidelines for Pleural Disease, PI was clinically addressed in this discussion as a case of CPPE or empyema and literature was searched accordingly (20).

Out of the 526 samples analyzed, 244 were positiveto culture; of which 110 cultures tested positive for Entero-bacteriaceae, indicating a significant number of negative results. These results could potentially be attributed to alternative etiologies or could be a result of previous antibiotic utilization (as 77.3% of our patient’s population have admittedly taken at least one antibiotic prior to sampling).

In the current study, E. coli, K. pneumoniae, and P. mirabilis were the main bacteria identified, indicating their significant role as causative agents of PI. These findings align with previous studies that have also identified the above-mentioned pathogens as the primary contributors to PI (25,26). Furthermore, the study revealed that those pathogens exhibited higher rate of resistance to third-generation cephalosporins, indicating their classification as ESBL phenotypes. The prevalence rates of ESBL reported in this study were significantly lower than a study from Burkina Faso, which reported 70% of ESBL-producing isolates in hospitalized patients (27). Genotypically, the most common ESBL gene was bla_TEM. This finding was also reported by Sonda et al (28). We did not detect bla_SHV gene.

The observed high resistance among the most common isolates to penicillin, quinolones, cephalosporins, and others is likely attributed to the higher utilization of those antibiotic classes in hospital settings. The existence of such association between antibiotic prescriptions and susceptibility pattern was previously highlighted in another hospital setting in Eritrea (29). Clinicians’ choice of broad-spectrum antibiotics or combination therapy may be suggestive of the infection being acquired in a hospital setting. It could be inferred that despite the treatment recommendations as per European Respiratory Society (ERS) and American Association for Thoracic Surgery (AATS), the selection of the treatment regimen must typically depend on infection setting, the local prevalence of microorganisms, and antibiotic resistance patterns (30,31).

Additionally, our study reported a high prevalence of MDR bacteria. This MDR rate aligns with previous study in Turkey reporting the widespread occurrence of MDR bacteria (26). The study findings revealed a higher prevalence of carbapenem resistance (7.2%) compared to the previous report by Dwomoh et al (32), which documented a resistance rate of 5.6%. Hackman et al (33) reported a similar rate of carbapenem resistance. However, our study observed significant disparities in the resistance rates of individual carbapenems (ertapenem, imipenem, meropenem) as well as the TZP combination compared to their study. These variances can be attributed to our study’s focus on the three predominant pathogens isolated (E. coli, K. pneumoniae, P. mirabilis). On the basis of available data, TZP and carbapenems appear to maintain significant effectiveness against the tested pathogens; the results were in accordance with those of a Malaysian observational cohort study (34). The recorded prevalence aligns with other studies conducted in Africa, such as those conducted in Nigeria (35) and South Africa (36). However, Egypt has recently reported a notably higher prevalence of carbapenem resistance (37).

The relatively low abundance of carbapenem resistance genes further supports this notion, indicating that the isolates in this study may not possess robust mechanisms of resistance to carbapenems. We did not detect cases of bla_KPC and bla_OXA-48 type of carbapenemase but 7.7% of bla_NDM-1. In 2022, a study investigating the global epidemiology of OXA-48-like β-lactamases, treatment option and pipeline development were conducted by Sara E. Boyd et al (38). The study found that the enzymes of bla_OXA-48 type are the most common carbapenemases among Enterobacteriaceae in much of western Europe. In Africa, the same study reported circulation of these types of β-lactamase in Tunisia, Algeria, Egypt, and South Africa. In West Africa, cases have been reported in Senegal and Nigeria. Mali did not provide data probably due to lack of adequate health care infrastructure and limited molecular diagnostic capabilities (38). Nabi Jomehzadeh et al found higher levels of bla_NDM-1 (31%) (39). In 2020, Muggeo et al reported the first description of bla_NDM-5 in Mali (18). Overall, β-lactam/β-lactam inhibitor combinations and carbapenems are suitable choices, as presented previously in observational studies and guidelines (40,41,42). However, it is crucial to interpret these results within the context of the provided data. The analysis is limited to the specific isolates and may not be representative of the broader population or other geographical regions.

According to this study, the presence of virulence factor genes in E. coli isolates was limited to a specific set. The occurrence of these factors varied, ranging from 2% for genes sta, slt, and lt, to 55.1% for the ipah gene. The ipah gene encodes Invasion Plasmid Antigen and is closely linked to immune system modulation in the host and bacterial survival. It is commonly detected in the majority of EIEC isolates (43).

The study has certain limitations. Firstly, it was conducted in a single center, which might restrict the applicability of the results to other populations or settings. Additionally, the sample size was relatively small, warranting larger multicenter studies to validate the findings. The study also lacked detailed information on patients’ antibiotic exposure history and prior hospitalizations, which could have influenced the prevalence of antibiotic resistance. Despite these limitations, the study emphasizes the significant prevalence of antibiotic resistance in the examined setting and underscores the ongoing need for surveillance and efforts in antibiotic stewardship to address this critical public health concern. Further research involving larger sample sizes, multicenter designs, and comprehensive patient data would greatly enhance our understanding and approach toward combating antibiotic resistance in our region.

In countries with limited resources such as those in West Africa, more specific socioeconomic and behavioral factors contribute to exacerbating this threat, among others: (i) certain common societal practices such as self-medication; (ii) a failing medical sector with insufficiently trained prescribers and inefficient diagnostic tools; or (iii) an uncontrolled drug chain with over-the-counter, improperly stored, counterfeit, and/or expired antibiotics favor the emergence of resistance. This study on the prevalence and characteristics of ESBL and carbapenemase-producing Enterobacteriaceae in pleural effusion in Mali holds great significance for low- and middle-income countries. By providing critical insights into the extent of resistance and molecular epidemiology, this research will facilitate the development of effective strategies to combat AMR, improve patient outcomes, and safeguard public health in Mali.

Acknowledgments

The authors would like to thank the managers of the molecular biology laboratories of the National Institute of Public Health and the University Clinical Research Center. The authors would also like to express their sincere gratitude to the Fogarty International Center for funding the registration of PhD student Aimé Césaire Kalambry through grant D43TW008652. Their support has been instrumental in the advancement of our understanding in this field and has contributed significantly to the successful completion of this manuscript.

Disclosures

Conflict of interest: The authors declare no conflict of interest.

Financial support: This research is part of a PhD study whose PhD program received financial support from the Fogarty International Center to cover the student’s registration fees only. No specific funds were allocated to this particular research project. The funding agency played no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.