Article Information

Issa NDIAYE*^{, 1, 7}, Bissoume Sambe BA⁹, Farma THIAM⁸, Mouhamadou Moustapha BOYE², Ousmane SOW¹, Abdoulaye CISSɹ, Babacar NDIAYE⁶, Thierno Abdoulaye DIALLO⁶, Cheikh FALL¹, Yakhya DIEYE¹, Baidy DIÈYE³, Assane DIENG², Amadou DIOP³, Guillaume CONSTANTIN DE MAGNY^{4, 5} and Abdoulaye SECK^6,7

¹Pôle de Microbiology, Institut Pasteur de Dakar, Sénégal

²Laboratoire de Bactériologie et Virologie, Hôpital Aristide Le Dantec , Dakar, Sénégal

³Laboratoire de Bactériologie et Virologie, Hôpital Albert Royer, Dakar, Sénégal

⁴MIVEGEC, Université Montpellier, CNRS, IRD, Montpellier, France.

⁵MEEDiN, Montpellier Ecology and Evolution of Disease Network

⁶Laboratoire de Biologie Médicale, Institut Pasteur de Dakar, Sénégal

⁷Faculté de Médecine, Pharmacie et Odontostomatologie, Université Cheikh Anta Diop, Dakar, Sénégal

⁸Laboratoire de Biologie Médicale, Hôpital régional de Pikine

⁹World Health Organization WCARO, Dakar, Senegal

*Corresponding author: Issa Ndiaye, Pole of Microbiology, Institut Pasteur Dakar, 36 Avenue Pasteur, Dakar, BP 220 Senegal.

Received: 01 May 2023; Accepted: 23 May 2023; Published: 07 June 2023

Citation: Issa NDIAYE, Bissoume Sambe BA, Farma THIAM, Mouhamadou Moustapha BOYE, Ousmane SOW, Abdoulaye CISSÉ, Babacar NDIAYE, Thierno Abdoulaye DIALLO, Cheikh FALL, Yakhya DIEYE, Baidy DIÈYE, Assane DIENG, Amadou DIOP, Guillaume CONSTANTIN DE MAGNY and Abdoulaye SECK. Antibiotic Resistance and Virulence Factors of Extended- Spectrum Beta-Lactamase-Producing Klebsiella Pneumoniae Involved in Healthcare-Associated Infections in Dakar, Senegal. Archives of Microbiology and Immunology. 7 (2023): 65-75.

Abstract

Keywords

Klebsiella pneumoniae, ESBL, antibiotic resistance, virulence genes, healthcare-associated infections

Article Details

1. Introduction

Antimicrobial resistance is a worldwide major public health problem, leading to elevated rates of illness and mortality, especially in immunocompromised patients [1]. Numerous reports and studies released by international organizations have outlined the far-reaching consequences of this issue on both healthcare and economic levels [2, 3].

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose-fermenting, facultative anaerobic and rod shape bacterium belonging to the Enterobacteriaceae family [4]. Klebsiella pneumoniae is also among the ESKAPE pathogens which adopt different mechanisms to “escape” from different antimicrobials actions [5] and is frequently associated with a wide range of healthcare associated infections (HAIs) such as pneumonia, bacteremia, urinary tract infection (UTI), soft tissue and burn infections [6]. Infections due to K. pneumoniae are difficult to treat because of combination of both expression of virulence factors and emergence of antibiotic resistances [4]. Some hypervirulent serotypes are particularly associated with various clinical syndromes characterized by community-acquired K. pneumoniae bacteremia with primary liver abscess, metastatic meningitis and endophthalmitis [7].

Klebsiella pneumoniae strains isolated from HAIs have exhibited a notable degree of antibiotic resistance and epidemiological investigations have identified multiple genes within these K. pneumoniae strains that confer resistance to commonly prescribed antibiotics including beta-lactams, fluoroquinolones, aminoglycosides, cyclines, macrolides, lincosamides, folate inhibitors, and phenicol [8]. In low and middle income countries, multidrug resistant K. pneumoniae is one important driver of unfavorable outcome in infections, primarily because suitable treatment options are often either unavailable or unaffordable [9].

Resistance of K. pneumoniae to antibiotics can be attributed to various mechanisms including modification of antibiotic target sites, alteration of metabolic pathways, activation of efflux pump systems, change in membrane permeability and release of antibiotic-inactivating enzymes [10]. Antibiotic resistance is a complex and multifactorial mechanism resulting from antibiotic exposure in hospital settings. Consequently, the selective pressure created leads to the development of numerous genetic mechanisms of resistance [11]. The acquired resistance over the years has led to the emergence of strains that are classified as Multi Drug-Resistant (MDR), Extensively Drug-Resistant (XDR) and Pan Drug-Resistant (PDR) strains [12].

Pathogenicity of K. pneumoniae can be attributed to the presence of virulence genes which encode for different types of virulence factors. These factors such as adhesin for attachment to host cells, capsules that are antiphagocytic, siderophores that aid the bacterium in its competition with the host for iron and other various endotoxins [13]. Virulence-associated genes searched includes those encoding regulators of mucoid phenotype A (rmpA), mucoviscosity associated gene (magA), uridine diphosphate galacturonate 4-epimerase gene (uge), type 3 fimbrae (mrKD), yersiniabactin receptor (fyuA) and the iron uptake system gene (kfu) which are responsible for colonization, invasion and pathogenicity, and have a predominant role in pathogenicity of K. pneumoniae strains isolated from HAIs [13, 14].

In Senegal, it is difficult to assess accurate incidence of healthcare associated infections due to limited research funds and a scarcity of published data. The few studies conducted in Senegal from 2005 to 2019 have shown a prevalence of healthcare associated infections ranging from 6.8% to 13.6% [15-19]. These studies have also identified highly resistant bacteria, including K. pneumoniae the most found one. However, none of them have investigated the molecular mechanisms of resistance. Therefore, the aim of this study is to investigate presence of key main virulence factors and the antibiotic resistance profiles of Extended-Spectrum Beta-lactamases (ESBL) strains of K. pneumoniae involved in HAIs in Dakar, Senegal, and the relationship between phenotypic and genetic patterns of antibiotic resistance. This is the first study investigating the molecular diversity of HAIs strains in Senegal.

2. Materials and Methods

2.1 Bacterial strains

In this retrospective study, we focused on 28 strains of K. pneumoniae isolated during routine laboratory activities from 2018 to 2021 of 2 major hospitals in Dakar, i.e. Hospital Aristide Le Dantec and the Center Albert Royer of Fann. Strains were isolated from various clinical specimens and were considered as hospital acquired if the infection occurs at least 48 hours after the hospitalization. As per international standard or university standard written ethical approval has been collected and preserved by the author(s).

The epidemiological data of collected strains were obtained from the registers at the bacteriology laboratory of each hospital. All laboratory techniques and procedures of isolation, identification, and storage of the included isolates in the current study were performed according to standards microbiological protocols included Gram staining, cultural characteristics on agar media and biochemical testing such as API 20E (Biomérieux, Marcy-l’Étoile, France) or Vitek2® system (BioMérieux® -France).

The identification of K. pneumoniae isolates was then confirmed using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS) (Vitek MS; BioMérieux, Inc., Marcy-l'Etoile, France) in Pasteur Institute of Dakar.

2.2 Antibiotic susceptibility testing

Nineteen (19) antibiotics disks (Bio-Rad) were selected from the standard list proposed by CA-SFM/EUCAST version 2022 and interpreted according to the given guidelines. The cyclins (tetracycline, minocyclin and doxycyclin) were interpreted from the CLSI guidelines, version 2020. Stored cultures of K. pneumoniae strains were subcultured in Bromocresol purple (BCP) incubated at 37° for 24 h. Inoculum were adjusted to 1.5×10⁸ CFU, corresponding to 0.5 McFarland and streaked on Mueller Hinton agar (Oxoid, UK) surface with sterile swab. Antimicrobial susceptibility was determined by strain growth zone diameter using the Kirby-Bauer method and interpretation done according to CA-SFM/EUCAST (version 2022) and CLSI guidelines (version 2021).

The presence of an extended-spectrum beta-lactamase (ESBL) was detected on the antibiogram by the synergy test based on the visualization of a "champagne cork" image between third- or fourth-generation cephalosporins and amoxicillin-clavulanic acid or piperacillin-tazobactam discs. The following 22 antimicrobials disks from Biorad were used: AMP: Ampicillin (10 μg), AMC: amoxicillin-clavulanic acid (20+10 μg), TIC: ticarcillin (75μg), TIM: ticarcillin-clavulanic acid (75+10 μg), FOX: cefoxitin (30 μg), CTX: cefotaxim (30 μg), CAZ: ceftazidime (30 μg), FEP: cefepime (30 μg), AZT: aztreonam (30 μg), IMP: imipenem (10 μg), ERT: ertapenem (10 μg), FOS: fosfomycin (10 μg), CIP: ciprofloxacin (5 μg), NOR: norfloxacin (10 μg), GN: gentamicin (10 μg), TMN: tobramycin (10 μg), AK: amikacin (30 μg), TET: tetracycline (30 μg), MIN: minocyclin (30 μg), DO: doxycyclin (30 μg), CHL: chloramphenicol (30 μg) and SXT: trimethoprim-sulfamethoxazole (1.25/23.75 μg).

Reference strain E. coli ATCC 25922 were used for quality control. Bacterial strains that were resistant to a minimum of at least 3 different classes of antibiotics were considered as MDR and those only susceptible to only one or two class of antibiotics and resistant to all sub classes in all classes of antibiotics were considered as XDR and PDR, respectively as previously described [12].

2.3 Detection of virulence and antibiotics resistance-associated genes by PCR

2.3.1 DNA extraction by thermolysis

Three colonies of each isolate on nutrient agar plate were picked and suspended in 200μl of distilled water. After vortexing, the cell suspension was boiled for 10 minutes and 150μl of the supernatant was collected after spinning for 10 minutes at 13,000 rpm in a microcentrifuge.

2.3.2 Determination of antimicrobial resistance genes

PCR was performed on Thermocycler 2720 (Applied Biosystems, Lincoln 113 Centre Drive, Foster City, California 94404 USA) to detect 26 antimicrobials resistance-associated genes include: β-lactamase genes (bla_TEM, bla_SHV, bla_CTX-M, bla_CTX-M-1 group, bla_CTX-M-2 group, bla_CTX-M-8 group, bla_CTX-M-9 group, bla_CTX-M-25 group and bla_CTX-M-15), carbapenem resistance-associated genes (IMP, NDM, OXA48, OXA23, VIM and KPC), quinolone resistance-associated genes (gyrA, gyrB, parC, parE, qnrA, qnrB, qnrC, qnrD and qnrS) and aminoglycoside resistance-associated genes (aac(6’)-Ib, AadA1).

Each reaction included positive and negative controls. The PCRs were carried out in 20 µl reaction volume (2.5 µl DNA + 17.5 Master Mix FIREPol® + 0.5µl of each primer). All primers used and PCR thermo cycling conditions in this study are listed in Table 5 and 6 respectively (Supplementary). PCR products were loaded on a 1.5% (w/v) agarose gel stained with ethidium bromide (0.5 mg/mL), electrophoretic migrations were done at 120 volts for 45 min in 1X TAE buffer and amplified fragments visualized using a GelDoc imager (BioRad).

2.3.3 Detection of virulence genes

Distribution of 6 genes (rmpA, mrKD, kfu, magA, uge, fyuA) associated with the virulence of K. pneumoniae were investigated. PCR were carried out with their specific primers and all PCR thermo cycling conditions are listed respectively in Table 7 and Table 8 (Supplementary).

Data were entered and analyzed using Excel.

3. Results

3.1 Total isolates

Twenty-eight (28) K. pneumoniae strains isolated from patients with HAIs were collected. Strains were isolated from 2018 to 2021 from different clinical samples: wound (n=14), urine (n=7), bacteriemia (n=7).

3.2 Antimicrobial susceptibility testing

Among the 22 antibiotics tested, antimicrobial susceptibility tests demonstrated that all the 28 strains were resistant to amoxicillin + clavulanic acid, ticarcillin + clavulanic acid, cefotaxim, ceftazidime, cefepime, aztreonam and cyclins (Table 1). Very high resistance rates (70-96.8%) were found for piperacillin + tazobactam, ciprofloxacin, norfloxacin, gentamicin, tobramycin, amikacin, trimethoprim/sulfamethoxazole. Ertapenem moderate resistance rates were found (32.1%) while a lowest level rate of resistance was found for imipenem (17.8%), cefoxitin (17.8%), chloramphenicol (25%) and fosfomycin (28.5%) (Table 1). Our work has shown ten carbapenem resistant strains in our cohort. Eighteen strains (64.3%) were considered as MDR as they were resistant to 1 antibiotic in 3 different classes and 10 strains (35.7) were XDR Table 4.

Table 1: Antibiotic Resistance of K. pneumoniae strains isolated from healthcare-associated

3.3 Gene diversity of antimicrobial resistance genes

The results of the current study showed that all of the strains harbored at least one ESBL gene. Specifically, 22 strains (78.5%) positives for bla_TEM, 21 (75%) for bla_SHV, and 21 strains (75%) for bla_OXA-1. All the strains were positives for bla_CTX-M with the variant bla_CTX-M15 identified in all of the 28 strains. For carbapenems, among the 10 resistant strains (35%), six (6) were found to carried OXA-48 gene and were phenotypically resistant to all antibiotics belonging to this class of antibiotics. The aac(6’)-Ib gene that confer resistance to aminoglycosides was found in 21 strains (75%). Regarding fluoroquinolone resistance, gene qnrB were detected for 16 strains (57.1%), qnrS for 11 strains (39.3%), gyrB for 3 strains (10.7%) and parE, qnrA, qnrC and qnrD for 1 strain respectively (3.6%). (Table 2 and Table 4).

Table 2: Genotypic resistance profile of 28 K. pneumoniae strains isolated from health-care associated infections

3.4 Molecular detection of virulence genes

Three virulence genes were detected in the 28 strains: uge (19, 67.8%), mrKD (21, 75%) and fyuA (13, 46.4%) (Table 3). Among the different virulence genes, we found mrKD gene alone in three strains (10.7%), association mrKD + fyuA in one strain (7.1%), association mrKD + uge in 7 strains (25%), and association mrKD + uge + fyuA in 11 strains (39.3%).

Table 3: Distribution of virulence genes amongst the K. pneumoniae from health-care associated infections.

Table 4: Characterization of K. pneumoniae isolated from healthcare-associated infections

AMC: amoxicillin-clavulanic acid, TCC: ticarcillin-clavulanic acid, FOX: cefoxitin, CTX: cefotaxim, CAZ: ceftazidime, FEP: cefepime, AZT: aztreonam, IPM: imipenem, ERT: ertapenem, MEM : meropenem, FOS: fosfomycin, CIP: ciprofloxacin, NOR: norfloxacin, GMN: gentamycin, TOB: tobramycin, AKN: amikacin, TET: tetracycline, MIN: minocyclin, DO: doxycyclin, CHL: choramphenicol, SXT: trimetroprim-sulfametoxazole

Table 5: Sequencing of primers used in PCR for 32 antimicrobials resistance-associated genes of K. pneumoniae

Table 6: Thermo cycling conditions of PCR for 32 antimicrobials resistance-associated genes of K. pneumoniae

Table 7: Sequencing of primers used in PCR for 6 virulence-associated genes of K. pneumoniae

Table 8: Thermo cycling conditions of PCR for 6 virulence-associated genes of K. pneumoniae

4. Discussion

The aim of this study was to investigate the distribution of virulence genes, phenotypic and genotypic patterns of antibiotic resistance among ESBL strains of K. pneumoniae isolated from HAIs from 2018 to 2021 in two hospitals (Dantec and Albert Royer) in Dakar, Senegal. In our study, all K. pneumoniae isolates showed MDR patterns displaying high rates of resistance to commonly used antibiotics for treating K. pneumoniae infections, such as β-lactams (100%), aminoglycosides (82.1%), fluoroquinolones (96.4%), cyclins (100%), Fosfomycin (28.5%) and trimetroprim-sulfamethoxazole (92.8%). A worldwide meta-analysis with 47 studies estimated the prevalence of antibiotic resistance in healthcare-associated MDR K. pneumoniae [20]. According to this meta-analysis, the resistance rates to various classes of antibiotics were as follows: β-lactams (91.5%), aminoglycosides (85.1%), quinolones (87.2%), cyclins (34%), sulphonamids (51%), polymyxins (14.9%) and other classes of antibiotics (38.3%).

At the molecular level, the observed high resistance to β-lactam antibiotics can be attributed to the expression of bla_TEM and bla_SHV genes that are expressed in 92.8% of the tested strains. Among these strains, 17 (60.7%) carried both bla_SHV, bla_TEM and bla_CTX-M genes. Specifically, bla_CTX-M15, belonging to the bla_CTX-M1 group, was the only variant identified in our study and is known to be widely distributed globally [21]. The CTX-M groups and bla_SHV are the major ESBLs phenotype worldwide [22] included in HAIs [23]. The bla_CTX-M genes are predominantly plasmid-encoded [24] and are the predominant β-lactamase conferring resistance in K. pneumoniae strains and other gram-negative bacterial to new broad spectrum β-lactam antimicrobials.

Ten K. pneumoniae strains were resistant to ertapenem and out of those, six also showed resistance to meropenem and imipenem. In Senegal, different studies have shown variable resistance rate to imipenem in healthcare-associated strains from 6.7 to 11.2% [18, 25, 26] and up to 72.2% [27]. The carbapenemase gene OXA-48 was detected in 6 strains while it was not found in the four strains that were only resistant to ertapenem. Further investigation may be needed to identify the mechanisms behind this resistance phenotype. ESBLs genes can also enable bacteria to resist to other classes of antimicrobials and 82.14% and 92.85% of the studied strains are respectively resistant to at least one antibiotic in aminoglycosides and fluoroquinolones family. Twenty (20) strains were resistant to amikacin, 27 (96.4) to ciprofloxacin and 23 (82.1) to norfloxacin.

Three studies conducted in Aristide Le Dantec hospital (Senegal) have investigated the prevalence of antibiotic resistance in healthcare-associated infections (HAIs). The results of these studies have demonstrated varying levels of resistance to aminoglycosides and ciprofloxacin. Specifically, the resistance rates for aminoglycosides were reported as 27%, 80%, and 84.6%, while the resistance rates for ciprofloxacin were found to be 55%, 81.43%, and 87% [18, 26, 28]. Two others studies conducted in the neurosurgery service of Fann Hopsital (Senegal) and the urologic service of Aristide le Dantec hospital have shown prevalence for amikacin resistance respectively of 16.7% [25] and 44.5% [27]. Although it has been used for several years, resistance for fosfomycin and chloramphenicol has remained low inKlebsiellaspp[29] compared to our study. We found respectively 8/28 (28.5%) and 7/28 (25%) resistant strains for fosfomycin and chloramphenicol antibiotics. Genes encoding quinolone plasmid-mediated resistance qnrB, qnrS and aminoside resistance gene aac(6’)-Ib were prevalent at high rates (75%) and are mostly responsible for resistance to fluoroquinolones and aminoglycosides, in concordance with some recent studies [24, 30].

Among the virulence factors investigated, three out of the six targeted genes were detected in the K. pneumoniae strains, with varying proportions. The mrKD gene encoding for a type 3 fimbriae (adhesins) was found in 21/28 (75%) of the studied isolates and is involved in the adhesion to epithelial, urinary and respiratory cells and promote biofilms development [14]. This finding aligns with the well-established ubiquitous nature of these fimbriae in K. pneumoniae [31]. The fyuA gene was found in 13/28 strains (46.4%), encoding for a yersiniabactin receptor, one of the most upregulated gene in biofilm formation specially in iron-poor environments such as human urine [32]. Additionnaly, the uge gene was found in 19/28 strains (67.8%) and codes for uridine diphosphate galacturonate 4-epimerase, express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface of the bacteria. Therefore, this gene is essential for K. pneumoniae virulence and strains that carried this gene were more virulent [33].

Our research findings demonstrate the existence of multidrug-resistant strains that owe their resistance to various genes within their genome. The combination of these genes, at times, contributes to an elevated resistance level against different antibiotics. Moreover, specific virulence factors are present, and their expression has the potential to escalate the severity and/or duration of infections. A sequencing of these strains should provide a better understand of the clones all the factors of virulence and resistance present in these strains and occurrence of mobile genetic elements.

5. Conclusion

The emergence of multidrug resistant K. pneumoniae as a significant aetiologic agent in HAIs poses a growing public health concern not only for Senegal but all over the world. The successful spread of these multidrug resistant K. pneumoniae has been largely facilitated by the fact that K. pneumoniae itself is a notorious healthcare associated pathogen leading to outbreaks in hospital seetings, in addition to other factors, such as inappropriate use of antibiotics , high-density populations, poor infection control, international travel and medical tourism. This study highlights the need to establish a comprehensive antimicrobial resistance surveillance network in Senegal for K. pneumoniae and other major healthcare-associated bacteria. Such a network would enable monitoring of emerging resistance trends and novel resistance mechanisms within hospitals. We also recommend strengthening antimicrobial stewardship rules and implementing robust infection control measures in healthcare facilities to mitigate the selective pressures that drive the emergence and spread of multidrug-resistant strains. By adopting these strategies, we can proactively combat antimicrobial resistance and safeguard patient health in hospital settings.

Ethical Research Approval

As per international standard or university standard written ethical approval has been collected and preserved by the author(s).

Consent for publication

All authors have given their consent for this publication.

Competing interests

The authors have not declared any conflict of interest.

Author’s contributions

MMB, AC, FT and OS helped to collect samples and data. MMB, AD, AD were involved in strains isolation and to perform some antibiogram susceptibility testing and contributed to antimicrobial resistance profiles interpretation. BN, FT, CF and YD helped revised the manuscript. BSB, GCDM, AS designed the study, supervised the results analysis and interpretation, and revised the manuscript. NI is the main author hence, participated to the experiment design, performed the lab work, did the bibliographic review, analyzed and interpreted the results, designed the figures and drafted the manuscript.

All the authors have read and approved the submitted version of the manuscript.

Funding

This study was supported by Institutional funds from GCDM at Institut de Recherche pour le Développement, France

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