available online on 15.08.2024 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright   © 2024 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                            Research Article

Phenotypic characterization and antibiotic resistance of enterobacteria strains isolated from samples of patients in the towns of Moundou and Sarh in Chad

Kadidja Gamougam¹, Mayoré Atéba Djibrine²*, Abdoulahi Ousman Hissein³, Abdelsalam Tidjani³

¹ Laboratoires du Centre Hospitalier – Universitaire de Référence Nationale (CHU-RN) de N’Djamena-Tchad. BP 130

² Institut National Supérieur du Sahara et du Sahel d’Iriba, Tchad

³ Faculté des Sciences de Santé Humaine, Université de N’Djamena, P.O. Box 1117, N'Djamena-Tchad

 

 

Introduction

Consumption of antibiotics kills the bacteria that cause disease and also kills those that protect the body against infections1. According to Public Health France, at the molecular level, resistance to antibiotics results either from chromosomal mutations (modification of genes already present) or from the acquisition of plasmids which are transmitted from bacteria to bacteria². Plasmid resistance is the most widespread (80% of acquired resistance) and can concern several antibiotics, or even several families of antibiotics, in which case we speak of multi-resistance².

In the United States, infections caused by antibiotic-resistant germs are difficult and sometimes impossible to treat³. Beta-lactams (mainly cephalosporins and extended-spectrum carbapenems) and fluoroquinolones are the main therapeutic choices to treat infections caused by these microorganisms⁴. However, resistance to these compounds has been reported more and more frequently in Europe in recent years^4,5. 

The main causes associated with this antibiotic resistance are overuse, inappropriate prescription, extensive agricultural use as well as the regulatory obstacle to the development of new antibiotics. Unlike developed countries, in developing countries, antibiotics are not regulated and can be found over the counter^6,7.

The impact of antibiotic resistance has health and economic consequences^8,9. In sub-Saharan Africa, the mortality rate linked to antimicrobial resistance is estimated at 27.3 deaths per year per 100,000 inhabitants⁹.

In Chad, some work carried out by independent researchers revealed strong resistance in a wide variety of sample types^{10, 11, 12, 13, 14, 15, 16}.

The objective of this study is to identify the strains of enterobacteria coming from samples of patients admitted to the Moundou and Sarh health centers, and to report their resistance profiles.

MatériAl AND Méthods

Period, setting and location of study

This prospective and transversal study, which took place in two stages, was conducted in two main cities in the south of Chad, namely Moundou and Sarh. 

A first stage took place from September to December 2021, and related to the collection of samples, their cultivation and conservation of the strains in the provincial hospitals of the respective cities.

A second stage which was carried out from February to April 2022, concerned the transport of the strains to the laboratory of the National University Reference Hospital Center (CHU-RN) of N’Djamena, their awakening, their identification and the antibiogram.

 

 

Figure 1: Geographic location of the study sites (Jean-Pierre Gami et Hassan Nadjara Kanika, 2022)

 

Sampling 

The study involved 278 samples from 2 types of samples taken from hospitalized or non-hospitalized users were collected from 133 urines and 145 stools from patients of all ages in the pots and cultured them.

Isolation and identification of bacteria

The culture was carried out on CLED (Citrate Lactose Electrolyte Deficient) agar media for urine, Mac Conkey supplemented with ceftriaxone for stools. All strains preserved in Trypcase soy agar were brought back to the CHU-RN laboratory for reisolation, identification and antibiogram.

Morphological identification by Gram staining and biochemical identification by the oxidase test and using API 20E galleries from Biomérieux and Enterosystem 18R. The digital profile of the strain was established and interpreted according to the API 20E analytical catalog (bioMérieux, France) or Enterosystem 18R from Liofilmchem to determine the name. 

Antimicrobial susceptibility testing

The antibiogram was carried out by the diffusion method or Kirby Bauer by flooding with an inoculum at the Optical Density (OD) of 0.5 McF on Muller Hinton medium with 14 antibiotic disks in 120 square Petri dishes. mm. This method was used for all strains. The interpretation of the diameters as Sensitive (S), Intermediate (I) or Resistant (R) was done in accordance with the standards of the Antibiogram Committee of the French Society of Microbiology¹⁷. The reading was done on the basis of the inhibition diameters. E. coli ATCC 25922 was used as a control strain. The antibiotics used were: Beta-Lactamines Ampicillin (10µg), Amoxicillin + Clavulanic Acid (10µg), Ticarcillin (75µg), Cefalotin (30µg), Cefotaxime (30µg), Ceftriaxone (5µg), Imipenem (10µg), Meropenem (10µg )); Aminosides Gentamicin (30 μg, 10 μg), Amikacin (30 μg)); Quinolone and Fluoroquinolones Nalidixic acid (30 μg), Ciprofloxacin (5 μg), Levofloxacin (5 μg)); Sulfonamides, Trimethoprim-sulfamethoxazole (1.25/23.75µg)). 

Resistance phenotypes to β-lactams, aminoglycosides, quinolones, and various antibiotics were determined for all strains. The strains were subjected to the synergy test for the search for ESBL according to the recommendations of EUCAST (2017)¹⁸. The synergy test was carried out by placing the AMC and C3G (ceftriaxone, cefotaxime, cefixime) or aztreonam discs 3 cm apart. The presence of ESBL was revealed by the appearance of synergy between the discs giving a so-called “champagne cork” appearance.

Data processing Statistical analyzes of data

The data collected was entered into Excel and analyzed using Statistical Package for the Social Sciences (SPSS) and Excel software. Differences were considered significant when p < 0.05.

Results

Prevalence of enterobacteria

A total of 278 samples were collected in the 2 hospitals in cities located in the south of the country (see Figure 1), including 145 stools and 133 urine. The results are presented in Tables 1.

Of the 278 samples (urine and stool combined) analyzed, the overall prevalence of enterobacteria was 39.92%. Of the 111 strains of enterobacteria isolated, we note a predominance of Escherichia coli (n=55) followed by the genus Enterobacter (n=17) and klebsiella (n=11). 

The Salmonella genus is found mainly in Moundou (8/9) as is the Serratia genus in Sarh (n=7). The genera Citrobacter (n=6) and Shigella (n=2) are identified only in Sarh and the genus Proteus (n=4) only in Moundou (Table 2).

 

 

Table 1 :  Distribution of germs by sample type

Germs	Sample type		Total
	Stools (n = 145)	Urine (n = 133)
Citrobacter freundii	00	06	06
Enterobacter Spp	09	08	17
Escherichia coli	34	21	55
Klebsiella Spp	07	04	11
Proteus mirabilis	02	02	04
Salmonella Spp	07	02	09
Serratia Spp	02	05	07
Shigella flexneri	00	02	02
Total	61	50	111

 

Table 2: Distribution of Enterobacteria by site

Germs	Collection site		Total	Proportion (%)
	Moundou	Sarh
Citrobacter freundii	00	06	06	2.15
Enterobacter Spp	11	07	17	6.11
Escherichia coli	26	29	55	19.78
Klebsiella Spp	03	08	11	3.95
Proteus mirabilis	04	00	04	1.43
Salmonella Spp	08	01	09	3.24
Serratia Spp	01	06	07	2.52
Shigella flexneri	00	02	02	0.72
Total	53	59	111	39.92

 

 

Enterobacteriaceae resistance phenotypes

The samples of 2 pathological products are examined by culture, biochemical identification and antibiogram carried out with selected antibiotic disks. The following results are observed :

In Sarh as well as in Moundou, the germs identified have varying sensitivity to the antibiotics tested. The overall resistance to aminopenicillins and 1st generation cephalosporins for the 2 cities is between 91 and 98%. For 3rd generation cephalosporins, the resistance rate is generally higher in Sarh than in Moundou; but remains the same in the 2 cities 75.8% in Sarh and 80.7% in Moundou for Escherichia coli with ceftriaxone, an extended spectrum beta-lactamase. No carbapenemase in Moundou, but in Sarh a strain of Salmonella resistant to imipenem. The strains have reduced sensitivity to quinolones and fluoroquinolones, much more resistance to nalidixic acid on average 78% for the 2 cities for Escherichia coli. Salmonella, Shigella and Serratia strains have good sensitivity (100%) to fluoroquinolones and aminoglycosides in Sarh, whereas in Moundou the salmonella strains are 62.5% resistant to ciprofloxacin and 37.5% to levofloxacin. A sensitivity to aminoglycosides of around 40%, but Amikacin still remains a little more active than gentamicin. Trimethoprim/Sulfonamides remain the least active antibiotic of all the strains identified. They are even 100% resistant to Escherichia coli, Klebsiella, Enterobacter, Serratia (K.E.S), Shigella and Salmonella in Sarh and to Escherichia coli, Klebsiella, Serratia and Proteus in Moundou. Salmonella at 87.5% and Enterobacter at 90.9% in Moundou.

In addition to betalactamase, some strains have associated resistance to fluoroquinolones and aminoglycosides for strains of Escherichia coli, Klebsiella and Enterobacter.

The resistance phenotypes of enzyme-producing Enterobacteriaceae are diverse, ranging from penicillinase (high and low level), cephalosporinase, extended spectrum beta-lactamase (ESBL) through multi-resistant bacteria to carbapenemase.

 

 

 

Table 3: Resistance phenotype of germs isolated in Moundou

Germs                             Antibiotics	Entero spp  (n = 11)	E. coli  (n = 26 )		Kleb spp (n = 3)				Proteus spp (n = 4 )		Salmo.  (n = 8)		Serrat. spp (n = 1 )
	R        S		R         S			R        S			R         S		R        S		R        S
Beta-Lactams
Ampicillin	11      00		26      00			03      00			04       00		07       01		01       00
Amoxicillin + Clavulanic acid	11      00		26         00			03      00			04       00		08       00		01       00
Ticarcillin	11     00		26      01			03      00			01       03		08      00		01       03
Cefalotin	11     00		26      00			03      00			04       00		08      00		01       00
Cefotaxime	08     03		21       05			01      02			02       02		04      04		01       00
Ceftriaxone	02     09		05     21			01      02			02       02		06      02		00     01
Imipenem	00      11		00      26			00      03			00       04		00      08		00     01
Meropenem	00      11		00      26			00      03			00      04		00      08		00     01
Quinolone and Fluoroquinolones
Nalidixic acid	08      03		21      05			01       02			03      01		05     03		00     01
Ciprofloxacin	05     06		21      05			01       02			03      01		05     03		00     01
Levofloxacin	05      06		21       05			01       02			03      01		03     05		00     01
Aminoglycosides
Gentamicin	06      05		14       12			01       02			03     01		02     06		00     01
Amikacin	07      04		09        17			01       02			03      01		00     08		00     01
Sulphonamides
Trimethoprim-sulfamethoxazole	10      01		00       26			03       00			04     00		07     01		01     00

R = Resistant ; S = Sensitive ; Entero. spp = Enterobacter Spp ; E. coli = Escherichia coli ; Kleb. spp = Klebsiella spp ; Proteus m. = Proteus mirabilis ; Serrat.spp. = Serratia spp ; Salmo.. = = Salmonella

Table 4 : Resistance phenotype of germs isolated in Sarh

Germs   Antibiotics	Citrobac. f.  (n = 6)		Entero. spp  (n = 6)		E. coli  (n = 28)		Kleb. spp  (n = 7)		Salmo. (n = 1)		Serrat. spp.  (n = 3)		Shige. f.  (n = 2)
	R	S	R	S	R	S	R	S	R	S	R	S	R	S
Beta-Lactams
Amoxicillin	06	00	06	00	28	00	07	00	01	00	03	00	02	00
Ampicillin	06	00	06	00	27	01	07	00	01	00	02	01	02	00
Amoxicillin + Clavulanic acid	06	00	06	00	27	01	07	00	01	00	02	01	02	00
Ticarcillin	06	00	06	00	27	01	07	00	01	00	00	03	02	00
Cefalotin	06	00	04	02	28	00	07	00	01	00	03	00	02	00
Cefotaxime	02	04	02	04	21	07	05	02	01	00	00	03	01	01
Ceftriaxone	02	04	02	04	21	07	06	01	01	00	00	03	01	01
Imipenem	00	06	00	06	02	26	01	06	01	00	00	03	00	02
Meropenem	00	06	00	06	02	26	01	06	01	00	00	03	00	02
Quinolone and Fluoroquinolones
Nalidixic acid	02	04	02	04	21	07	05	02	01	00	00	03	01	01
Ciprofloxacin	02	04	02	04	19	09	05	02	00	01	00	03	00	02
Levofloxacin	02	04	02	04	19	09	03	04	00	01	00	03	00	02
Aminoglycosides
Gentamicin	02	04	02	04	13	15	04	03	00	01	00	03	00	02
Amikacin	02	04	02	04	13	15	04	03	00	01	00	03	00	02
Sulphonamides
Trimethoprim-sulfamethoxazole	04	02	06	00	28	00	07	00	01	00	03	00	02	00

R = Resistant ; S = Sensitive ; Citrobac. f. = Citrobacter freundii ; Entero. spp = Enterobacter Spp ; E. coli = Escherichia coli ; Kleb. spp = Klebsiella spp ; Salmo. = Salmonella ; Serrat.spp. = Serratia spp ; Shige. f. = Shigella flexneri. 

 

Discussion

In the two cities in the south of the country, namely Moundou and Sarh, out of the 278 samples analyzed, 111 strains were isolated. The results obtained showed a prevalence of enterobacteria of 39.92%, these results are higher than those of Hamadou and al. in 2023¹⁶, 21%. The genus Escherichia coli 49.55% (55/111) is predominant followed by Enterobacter spp 15.31%, Klebsiella spp 9.91%, Salmonella spp 8.11% and Serratia spp 6.30%, a superposable result to several studies on enterobacteria. In Sarh (50%) and Moundou (49.05%) of Escherichia coli, results lower than those of N’Djamena, Yandai et al., in 2019¹³ (64.5%), Hamadou et al. 2023¹⁶, 74%, Ouchar et al. in 2019¹², 63.83% and 21.28%, Djimabi et al. 63.93% in Togo¹⁹, but comparable to those of Ebongue et al. 2015 (Douala)⁵ Escherichia coli 48.5%.

The overall resistance to aminopenicillins for the 2 southern cities is above 90% (91-98%), a result similar to that of Yandai et al. (96.66-100%) in 2019 in Chad¹³. Amoxicillin associated et the beta-lactamase inhibitor such as clavulanic acid is inhibited by 98.30% in Sarh and 100% in Moundou, results well above those of Santos et al. (62.85%) in July 2020 in Brazil²⁰. 

Resistance to 3rd generation cephalosporins is present in a large number of enterobacteria. In Sarh, Escherichia coli has a resistance to ceftriaxone of 64.4% and 68% in Moundou, a result beyond that observed by Cardinale et al. (41.3%) in the population of N’Djamena²¹ (Cardinale et al., 2020).

These results are consistent with those of Ouchar et al. (2019)¹² which confirm that betalamase-producing enterobacteria are mainly Escherichia coli, i.e. 71.9%, and Klebsiella pneumoniae (16/89). An extended spectrum beta-lactamase developed by all strains, these results confirm those of Da et al. (2022) taking stock of resistance in Sub-Saharan Africa, the results can reach 80% in community infections²².

In Sarh a Salmonella strain resistant to imipenem and 3rd generation cephalosporins, resistant to nalidixic acid but sensitive to fluoroquinolones and aminoglycosides, a strain with a resistance profile different from those identified in N'Djamena in 2019 by Hamadou et al. (2017)¹⁶ which are resistant to ciprofloxacin (80%) and gentamicin (80%). Unlike Sarh where the carbapenemase rate observed in Escherichia coli 7.14% and increasing according to the study by Ouchar et al. (Ouchar et al., 2019)¹², in Moundou, none are resistant to the carbapenems identified.

The data indicate a spread of multi-resistant bacteria to the main classes of antibiotics. In addition to betalactamase, certain strains have associated resistance (coreristance) to fluoroquinolones and aminoglycosides for Escherichia coli strains, 2 of which are resistant to all antibiotics tested. (7.14%), Klebsiella (66.66% for ciprofloxacin and 50% for gentmicin) and Enterobacter (33.33% for aminoglycosides and fluoroquinolones) in Sarh. In Moundou, Escherichia coli, in addition to resistance to C3G and fluoroquinolones (68.75%), co-resistance to gentamicin of 37.5%, a strain of Klebsiella ozanae multi-resistant to all antibiotics tested except carbapenems. these multi-resistances are also found in N’Djamena in 2019 by Hamadou et al. (2023)¹⁶.

The strains have reduced sensitivity to quinolones and fluoroquinolones, much more resistance to nalidixic acid at 75.86% in Sarh and 80.86% in Moundou for Escherichia coli. and for Klebsiella spp 33.33% in Moundou and 75% in Sarh. A rate higher than those reported by Yandai et al. in 2019 (31.66% - 43.47%)¹³, but lower than those of Ouchar et al. (91.67-100%)¹² in 2019 in N’Djamena. 

Strains of Salmonella, Shigella and Serratia have good sensitivity (100%) to fluoroquinolones and aminoglycosides in Sarh, whereas in Moundou the strains of salmonella are 62.5% resistant to ciprofloxacin, rates lower than those of Hamadou et al. 80% between 2017-2019¹⁶ and Ouchar et al in 2019, 90.28 for ciprofloxacin and 80.85 for levofloxacin¹². A result above that of Guillard et al in 2020²³.

Reduced sensitivity to aminoglycosides, approximately 40% resistance to gentamicin compared to 73.61 for Ouchar et al. in 2019¹², 33.96% for Amikacin remains a little more active in Moundou, a rate higher than those of Ouchar et al. in 2019 13, 15.28 for amikacin.

Trimethoprim - sulfamethoxazole remains the least active antibiotic of all because Escherichia coli, Klebsiella, Enterobacter, Serratia (K.E.S), Shigella and Salmonella in Sarh and Escherichia coli, Klebsiella, Serratia and Proteus in Moundou are 100% resistant to Trimethoprim-sulfamethoxazole. Salmonella at 87.5% and Enterobacter at 90.9% in Moundou, a rate comparable to that reported by Yandai et al. (65% - 95.65%) in N’Djamena in 2019¹³.

Conclusion

The enterobacteria identified in urine and stools in Moundou and Sarh are mainly Escherichia coli followed by the K.E.S. group. This result is consistent with several other studies carried out around the world and in Chad, particularly in N’Djamena.

In terms of sensitivity to antibiotics, our work confirms that certain enterobacteria tested are multi-resistant to the different antibiotics used. These different resistances are mainly due to self-medication, poor compliance with instructions and inadequate prescriptions. This situation is a real challenge to initiate mass awareness-raising on the risks linked to bacterial resistance. The control of multi-resistant enterobacteria strains in the environment through the strengthening of technical capacities for their detection must receive close attention from the competent authorities.

Acknowledgement

The authors sincerely thank the general management and all the staff of Hospital of Moundou and Hospital of Sarh for their multifaceted support and all the staff of the National Reference Teaching Hospital. We would also like to thank the International Health Support Center for its material support and finally we thank the Solidarity Fund for Innovative Projects for its financial support during the Laboratory analyses.

Conflicts of interest

The authors declare that they have no competing interests.

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