ABSTRACT
Pseudomonas aeruginosa is generally susceptible to antibiotics of the families of Beta-lactam, aminoglycosides and quinolones. The aim of this study was to evaluate the antimicrobial susceptibility of P. aeruginosa strains in Bamako, Mali. P. aeruginosa strains were isolated on Drigalski agar. Antimicrobial susceptibility testing was performed using the disc diffusion method on Mueller-Hinton agar. Among 317 non repetitive strains recovered from 2010 to 2019, there were 246 (77.6%) hospital strains and 71 (22.4%) extra-hospital strains. Colistin (100%), imipenem (98.4%), ceftazidime (89.3%), amikacin (85.2%) and piperacillin (72.3%) were the most active antibiotics against our P. aeruginosa strains. Of the strains 11 (3.5%) were multi-drug resistant (MDR) and 5 (1.6%) were extensively drug-resistant (XDR). The extra-hospital P. aeruginosa strains were more susceptible to aztreonam (91.5% vs 60.6%; P = 0.0000018), piperacillin (84.5% vs 68.7%; P = 0.013), gentamycin (84.5% vs 62.2%; P = 0.00071), netilmicin (56% vs 32.5%; P = 0.0045) and ciprofloxacin (79% vs 65.4%; P = 0.0455) than the hospital strains. Colistin, imipenem, ceftazidim, amikacin and piperacillin have a high-level activity against P. aeruginosa in Bamako.
Key words: Pseudomonas aeruginosa, antimicrobial susceptibility, Bamako, Mali.
Pseudomonas aeruginosa is a strictly non-fermenting aerobic Gram-negative bacillus that belongs to the Pseudomonadaceae family. P. aeruginosa is involved in various infections: urinary tract infections, abscesses, bacteremia, pulmonary infections, bone and joint infections, eye infections, infections of the otolaryngological sphere, meningeal infections, skin infections, enteritis, and endocarditis Avril et al., 2000; Wu et al., 2015). P. aeruginosa which is catalase and oxidase-positive, generally produces two pigments: pyocyanin (blue-green) and pyoverdine (yellow-green and fluorescent), Avril et al., (2000). P. aeroginosa is susceptible to carboxypenicillins, ureidopenicillins (mezlocillin, piperacillin), some 3rd generation cephalosporins (cefsulodine, cefoperazone, ceftazidime, cefepime, and cefpirome), carbapenems (imipenem), monobactams (aztreonam), certain aminoglycosides (gentamicin, netilmicin, and amikacin), fluoroquinolones, and colistin (Avril et al., 2000). P. aeruginosa has a natural resistance to aminopenicillins, 1st and 2nd generation cephalosporins, cefotaxime, cotrimoxazole, tetracyclines, chloramphenicol and nalidixic acid (Avril et al., 2000).
In addition to this natural resistance, there are acquired resistances which constitute the whole problem with this bacterium, particularly in hospitals and increasingly in community settings. These acquired resistances could be found in β-lactam (cephalosporin), aminoglycosides, fluoroquinolones (Avril et al., 2000; Kouamé et al., 2016; Weldaghen et al., 2003).
In Mali, there are very limited data on the susceptibility of P. aeruginosa to antibiotics, and given the increasing antibiotic resistance worldwide, the aim of this study was to evaluate the susceptibility of P. aeruginosa to antibiotics in Bamako.
Study site and setting
This was a retrospective study carried out in the Medical Biology and Hospital Hygiene Laboratory of the University Teaching Hospital of the Point G, Bamako, Mali from January 1st, 2010 to December 31st, 2019. The University Teaching Hospital of the Point G is the third-pyramidal reference in Mali, and has 522 beds divided between the surgical, intensive care and medical departments.
Bacterial strains
While patients admitted to the different departments of the University Teaching Hospital of the Point G, were hospitalized patients (in-patients), those who were coming to the hospital, for medical consultation, laboratory tests and/or X-Ray were not hospitalized and were called out-patients.
The hospital strains of P. aeruginosa were isolated from samples from hospitalized patients at the Point G University Teaching Hospital.
The extra-hospital strains of P. aeruginosa (community strains) were isolated from samples from out-patients.
The 317 non-repetitive strains isolated from samples collected from in-and out-patients visiting the University Teaching Hospital of the Point G. The strain isolated was done on Drigalski agar (Bio-Rad, France) at 37°C.
The identification of the strains was made either on the production of pyocyanin and pyoverdine on respectively King A and King B (Bio-Rad, France) media, on oxidase (Bio-Rad, France) and catalase (bioMérieux, France) positive reactions, and by the API 20 NE systems (bioMérieux, France).
Susceptibility to antibiotics test
The antimicrobial susceptibility testing was carried out on Mueller-Hinton agar (Bio-Rad, France) by the disc method (Agar diffusion method). The strains of P. aeruginosa were classified as "susceptible", "intermediate" or "resistant" according to the recommendations of the Antibiotic Committee of the French Society of Microbiology/European Committee on Antimicrobial Susceptibility Testing in 2015 (CA-SFM/EUCAST) (Jehl et al., 2015). The strains of P. aeruginosa classified as <<intermediate>> to the antibiotics tested were considered resistant.
Laboratory procedure
The antimicrobial susceptibility testing was performed on Mueller-Hinton agar (Bio-Rad, France) poured into a Petri dish. A colony isolated from an 18-24 h culture of P. aeruginosa was suspended in 5 ml of sterile saline solution which was calibrated to 0.5 MacFarland. Two drops of this suspension are then added in 10 ml of sterile distilled water. This second suspension is poured over the entire surface of the Mueller-Hinton agar poured into a Petri dish. The excess is poured into bleach. The seeded agar is left to dry for 15 min at 37°C inside the incubator. Please kindly note that the incubator is not used to dry seeded agar plate. Instead, the plates are allowed to stand on the bench for a while, before the antibiotic discs are introduced. After the seeded agar has dried, the blotting paper discs impregnated with the antibiotics to be tested are placed on the surface of the agar using a disc dispenser according to manufacturer’s instructions (Bio-Rad, France). After a first diffusion of the antibiotics in 30 min at room temperature, the Petri dish is incubated at 37°C for 18 to 24 h, in the inverted position (cover down). The reading is performed in measuring the diameter of inhibition of each antibiotic disc using a caliper in contact of growth.
Antibacterial agents tested
The antibiotics tested were ticarcillin (75 μg), piperacillin (75 μg), ceftazidime (30 μg), aztreonam (30 μg), imipenem (10 μg), gentamicin (15 μg), tobramycin (10 μg), netilmicin (30 μg), amikacin (30 μg), ciprofloxacin (5 μg), and colistin (50 μg) (Bio-Rad, France).
Multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes
The strains of P. aeruginosa intermediate or resistant to at least one molecule in three groups of antibiotics active against P. aeruginosa: (1) β-lactams except imipenem (ticarcillin, piperacillin, ceftazidime, and aztreonam), (2) imipenem, (3) aminoglycosides, and (4) ciprofloxacin were considered to be MDRs. The strains of P. aeruginosa intermediate or resistant to at least one molecule in each group of antibiotics have been considered as XDR (Barbier and Wolff, 2010; Magiorakos et al., 2012; Horcajada et al., 2019).
Ethics statement
The clinical specimens included in this manuscript were collected under public health surveillance of antimicrobial testing, and not as human subject research. Thus, submission to institutional review boards was not applicable. Participants were explained, and they consented to use the results. In addition, permission was received from Hospital Director for this manuscript.
Statistical analysis of data
The samples were collected under public health surveillance of antimicrobial resistance, and thus an estimated sample size was not previously determined. The data were entered and analyzed using Epi Info software 7.1 version. For the comparison of the results, we used the test of c² with a significance level P ≤ 0.05.
A total of 317 non-repetitive strains of P. aeruginosa were identified from 317 persons between 2010 and 2019. The mean age of patients was 48.77±18.5 years old, and the sex ratio (male/female) was 1:4 ratio.
The annual frequency of strains during the ten-year period is presented in Figure 1. Among these strains, 246 (77.6%) were of hospital and 71 (22.4%) of extra-hospital origin. The hospital strains were isolated in the wards of medicine (n = 185), surgery (n = 49) and intensive care unit (n = 12).
The distribution of P. aeruginosa strains according to the samples is shown in Table 1. The strains of P. aeruginosa have been isolated primarily from urine 143 (45.1%), pus 81 (25.6%), vaginal swabs 41 (12.9%), and/or sputum 26 (8.2%).
The antibiotic susceptibility of P. aeruginosa is reported in Figure 2. Thus, colistin, imipenem, ceftazidime, amikacin and piperacillin were the most active antibiotics against P. aeruginosa. Of the 317 strains 11 (3.5%) were MDR and 5 were XDR (1.6%). More specifically, 7 (2.8%) MDR-strains of P. aeruginosa were isolated in in-patients, and 4 (5.6%) in the out-patients setting, while 3 (1.2%) XDR-strains were isolated in in-patients, and 2 (2.8%) in the out-patients setting.
The susceptibility of antibiotics to P. aeruginosa strains isolated either from in- or out-patients is reported in Table 2. The strains isolated from out-patients were statistically more susceptible to aztreonam (P < 0.0000), piperacillin (P= 0.0130), gentamicin (P=0.007), netilmicin (P=0.0045) and ciprofloxacin (P=0.0455) than in-patients’ strains.
This study was carried out to evaluate the antimicrobial susceptibility of different P. aeruginosa strains isolated in our laboratory between 2010 and 2019. To the best of our knowledge, this study is the first study of its kind conducted in Bamako, Mali.
The identification of our strains of P. aeruginosa was based on their morphological and biochemical characteristics (Avril et al., 2000). The interpretation of the results was done with regard to international recommendations (CA-SFM/EUCAST) (Jehl et al., 2015).
This has public health implication in Mali as it was a surprise to find out that we have resistance to some antibiotics such as imipenem, ceftazidime, and amikacin which are not used in routine care. Thus, both in hospital area and outside hospital, the use of antibiotics should be guided by antimicrobial susceptibility testing results.
In this study, the strains of P. aeruginosa were of hospital and non-hospital origin, and the hospital strains were mainly from the medical and surgical departments. In Monastir in Tunisia, P. aeruginosa strains were isolated in intensive care, surgery and ear, nose, and throat (ENT) departments, mostly (Ben Abdallah, 2008).
This difference in the study site may explain the difference between the two studies.
In the present study, P. aeruginosa strains were isolated from different samples: urine, pus, vaginal samples, and blood cultures (Table 1), while in Monastir in Tunisia the strains of P. aeruginosa were isolated from pus (52.9%), respiratory samples (19.5%), urine (10.6%) and blood cultures (5%) (Ben Abdallah, 2008). The P. aeruginosa strains of Abdou-Souley Lié Moustapha (2002) were isolated in 2002 at the Point G University Teaching Hospital in the same samples as the present study. The sampling sites fit well with the pathogenicity of P. aeruginosa which determines various infections (Avril et al., 2000; Wu et al., 2015; Michel-Briand, 1992).
In Europe, 74% of P. aeruginosa strains were susceptible to ticarcillin, 80% to ceftazidime, 73% to aztreonam and 82% to imipenem (Rossolini and Mantengoli, 2008). Ticarcillin, ceftazidime and aztreonam were, respectively active in 46.9, 89.6 and 67.5% of the strains of P. aeruginosa.
Piperacillin was not active in 27.7% of the strains. The proportion of P. aeruginosa strains resistant to piperacillin varies from one country to another: it was 48.5% in Germany, 38.4% in France and 5.4% in the United Kingdom (Nordmann and Naas, 2012). This difference could be explained by the previous exposition to this antibiotic. Generally, this ureidopenicillin is not available in Mali. The susceptibility of the strains to imipenem is almost the same (Figure 2). The proportion of P. aeruginosa strains resistant to ceftazidime is 18.6% in France and 21.8% in Monastir in Tunisia (Ben Abdallah et al., 2008; Nordmann and Naas, 2012). This proportion was 10.4% in the present study (Figure 2).
The proportion of P. aeruginosa strains resistant to carbapenems (imipenem or meropenem) is 18.4% in France (Nordmann and Naas, 2012). In Monastir in Tunisia, the resistance rate of P. aeruginosa to imipenem was 19.6% (Ben Abdallah, 2008), while resistance to imipenem was 1.6% in the present study (Figure 2).
In this study the prevalence of P. aeruginosa strains MDR and XDR were low regardless of origin. Usually, the prevalence of MDR strains of P. aeruginosa varies from 15 to 30% in many regions (Horcajada et al., 2019). In 2017 in Spain, a multicenter study of P. aeruginosa infections found 26% of MDR strains and 17% XDR strains (Ben Abdallah, 2008). In the United States, out of 7,868 strains of P. aeruginosa isolated in 94 hospitals between 2013 and 2016, 1,562 (19.8%) were MDR and 717 (9.1%) XDR (Sader et al., 2017).
In 1990 at Henri Mondor Hospital in France, the resistance rate of P. aeruginosa strains to gentamicin and amikacin was 39.81 and 12.03%, respectively (Caron and Humbert, 1993). This rate is close to the present study with regards to gentamicin (Figure 2 and Table 2). This difference of resistance to specific and/or MDR strains could be explained by the low prevalence of P. aeruginosa and imipenem is rarely prescribed in setting of the present study.
The P. aeruginosa strains of Ben Abdallah et al. (2008). Ben Abdallah (2008) isolations in Monastir were more resistant to gentamicin (39.3%) as the present study. This strains of P. aeruginosa appear to be more resistant to amikacin than those from the Henri Mondor Hospital in France (Figure 2 and Table 2), and the strains of Ben Abdallah et al. (2008) isolated in Monastir, Tunisia, were resistant to amikacin at 19.2%.
In France, the P. aeruginosa resistance rate to ciprofloxacin was stable at around 25 to 30% according to Soussy (2012). The resistance rate of our extra-hospital strains to ciprofloxacin was identical to that of Soussy (2012) (Figure 2), and probably due to the baseline prescription of ciprofloxacin to many other bacterial diseases such as typhoid fever which is very common in the setting of the present study.
The strains of P. aeruginosa were susceptible to colistin (Avril et al., 2000), and the susceptibility of the strains of the present study to colistin was constant, because there was no resistance to this polymyxin (Figure 2).
This study has some limitations; first the data were retrospectively collected, and also were limited by the number of discs to be purchased for a complete full profile of antimicrobial resistance to the P. aeruginosa strains.
Despite these limitations, the study is unique as it collects consecutive strains of the P. aeruginosa isolated in both hospital and outside-hospital area in Bamako, Mali.
Colistin, imipenem, ceftazidime, amikacin and piperacillin were the most active antibiotics against P. aeruginosa. Ciprofloxacin and ticarcillin were active in every other strain in hospital area. The frequency of multidrug-resistant strains of P. aeruginosa is low in Bamako. Out-patients’ strains were more susceptible to aztreonam, piperacillin, gentamicin, netilmicin and ciprofloxacin than hospital strains.
We thank The Fogarty International Center and National Institute of Allergy and Infectious Diseases sections of the National Institutes of Health under award numbers U2RTW010673 for the West African Center of Excellence for Global Health Bioinformatics Research Training for their partially support of the study.
The authors have not declared any conflict of interests.
REFERENCES
|
Abdou-Souley Lié Moustapha FK (2002). Sensibilité et évolution de la résistance de Pseudomonas aeruginosa aux antibiotiques à l'hôpital du Point G [thèse]. Bamako : Université des Sciences, des Techniques et des Technologies de Bamako 67 p.
|
|
|
|
Avril JL, Dabernat H, Denis F, Monteil H (2000). Bactériologie clinique, 3ième édition. Paris: Ellipses 602 p.
|
|
|
|
|
Barbier F, Wolff M (2010). Multirésistance chez Pseudomonas aeruginosa : vers l'impasse thérapeutique. Médecine Science.
Crossref
|
|
|
|
|
Ben AH, Noomen S, Ben EK, Sahnoun O, Elargoubi A, Mastouri M (2008). Profil de sensibilité aux antibiotiques des souches de Pseudomonas aeruginosa isolées dans la région de Monastir. Médecine et Maladies Infectieuses 38(10):554-556.
Crossref
|
|
|
|
|
Caron F, Humbert G (1993). Aminoglycosides. Encycl Med Chir, Maladies Infectieuses 9 p.
|
|
|
|
|
Horcajada JP, Montero M, Oliver A, Sorli L, Luque S, Gomez-Zorrilla S, Benito N, Graue S (2019). Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clinical Microbiology Reviews.
Crossref
|
|
|
|
|
Jehl F, Lina G, Bonnet R, Bru JP, Caron F, Cattoir V (2015). Comité de l'Antibiogramme de la Société Française de Microbiologie/European Committee on Antimicrobial Susceptibility Testing.
|
|
|
|
|
Kouamé EC, Guessennd N, Mbengue Gbonon V, Konan F, Anne JC, Kacou N'Douba A, Dosso M (2016). Sensibilité aux antibiotiques des souches cliniques de Pseudomonas aeruginosa de 2005 À 2009 À Abidjan, Côte d'Ivoire. Revue Bio-Africa 15:33-38.
|
|
|
|
|
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JH, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice BL, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology Infections 18(3):268-281.
Crossref
|
|
|
|
|
Michel-Briand Y (1992). Infections à bacille pyocyanique. Encycl Med Chir, Maladies Infectieuses 14 p.
|
|
|
|
|
Nordmann P, Naas T (2012). β-lactamines et Pseudomonas aeruginosa. In. Courvalin P, Leclercq R (eds), Antibiogramme, 3ième édition. Paris: Eska pp. 189-206.
|
|
|
|
|
Rossolini GM, Mantengoli E (2008). Antimicrobial resistance in Europe and its potential impact on empirical therapy. Clinical Microbiology Infections 14(6):2-8.
Crossref
|
|
|
|
|
Sader HS, Castanheira M, Shortridge D, Mendes RE, Flamm RK (2017). Antimicrobial activity of ceftazidime-avibactam tested against multidrug-resistant Enterobacteriaceae and Pseudomonas aeruginosa isolates from U.S. medical centers, 2013 to 2016. Antimicrobial Agents and Chemotherapy.
Crossref
|
|
|
|
|
Soussy CJ (2012). Quinolones et bactéries à Gram négatif. In. Courvalin P, Leclercq R (eds), Antibiogramme, 3ième édition. Paris: Eska pp. 301-315.
|
|
|
|
|
Weldaghen GF, Poirel L, Nordmann P (2003). Ambler Class A extended-spectrum β-lactamases in Pseudomonas aeruginosa: novel development and clinical impact. Antimicrobial Agents and Chemotherapy 47(8):2385-2392.
Crossref
|
|
|
|
|
Wu W, Jin Y, Bai F, Jin S (2015). Pseudomonas aeruginosa. In. Tang YW, Sussman M, Liu D, Poxton Y, Schwarzman J (eds). Molecular medical microbiology, volume II. 2nd edition. New York, NY: Elsevier pp. 753-767.
Crossref
|
|
