In Vitro Activity of Ceftazidime/Avibactam Against Multidrug-Resistant Gram-Negative Bacilli in Niger

Abstract

Background:

The rise of multidrug-resistant Gram-negative bacilli (MDR-GNB) necessitates the evaluation of new antibiotics such as ceftazidime-avibactam (CZA). Data on its efficacy in Niger is scarce. This study assessed the in vitro activity of CZA against clinical MDR-GNB isolates.

Methodology:

This prospective cross-sectional study was carried out on 306 non-duplicate MDR-GNB isolates from a National Reference Laboratory from December 2023 to November 2024. Antimicrobial susceptibility testing was performed, and CZA resistance rates were determined. Statistical analysis identified factors associated with resistance.

Results:

Overall, 23.4% of MDR isolates were resistant to CZA. However, resistance varied dramatically by phenotype: 7.6% in ESBL-producing Enterobacterales (ESBL-E), 33.3% in carbapenem-resistant Pseudomonas aeruginosa (CRPA), 61.7% in carbapenem-resistant Enterobacterales (CRE), and 100% in carbapenem-resistant Acinetobacter baumannii (CRAB). Multivariate analysis confirmed that carbapenem resistance was the strongest predictor of CZA resistance (OR=19.00, 95% CI: 9.96-38.27, p<0.001).

Conclusion:

CZA demonstrates potent activity against ESBL-E in Niger, supporting its role as a carbapenem-sparing agent. However, the high resistance rates in CRE and CRAB preclude its empirical use for suspected infections with these pathogens. These findings underscore the critical need for routine CZA susceptibility testing and enhanced stewardship to preserve its utility.

Introduction

Antimicrobial resistance (AMR) is a major global health challenge in the 21st century, with multidrug-resistant (MDR) bacteria spreading rapidly across the world [1]. Sub-Saharan Africa bears a disproportionate burden, with an estimated mortality rate of 27.3 deaths per 100,000 population annually [2]. In this region, AMR has become one of the leading causes of mortality, surpassing malaria, HIV, and tuberculosis [3]. In Niger, the mortality rate attributable to AMR ranks 16th among 204 affected countries, with an estimated 7,100 deaths directly attributable to AMR and 31,300 deaths associated with it in 2019 [4].

Gram-negative bacilli (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and members of the Enterobacterales, are among the main pathogens responsible for MDR infections. Carbapenems remain the last-resort β-lactam antibiotics for treating infections caused by these organisms [5,6]. However, the increasing production of β-lactamases capable of hydrolyzing most β-lactam antibiotics, including carbapenems, has severely limited therapeutic options [7]. The misuse of antibiotics in human health, animal production, and environmental contamination has accelerated the global dissemination of resistant strains [8]. Consequently, the development of new therapeutic options has become an urgent priority [9].

Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination approved by the U.S. Food and Drug Administration in 2015 [10]. Avibactam acts by covalently but reversibly binding to β-lactamases, allowing it to inactivate enzymes such as extended-spectrum β-lactamases (ESBL), Amp C enzymes, and class A carbapénémases [11]. This combination restores the activity of ceftazidime against many MDR Gram-negative bacteria, including ESBL-producing Enterobacterales, carbapenemase-producing Klebsiella pneumoniae (KPC, OXA-48), and Pseudomonas aeruginosa with AmpC-type resistance [12].

Although several studies have evaluated the in vitro activity of CZA against MDR Gram-negative bacilli in different countries [13–15], data from Niger are lacking. This knowledge gap limits the ability to guide appropriate empirical therapy and antibiotic stewardship in the local context. Therefore, this study aimed to evaluate the in vitro activity of ceftazidime/avibactam against multidrug-resistant Gram-negative bacilli isolated from clinical specimens in Niger.

Methodology

Study design and period

This was a prospective cross-sectional study conducted on bacterial strains collected and stored at -80°C from December 2023 to November 2024.

Study population and sampling

The study population encompassed all MDR bacterial strains isolated from stored pathological products (urine, stool, pus, genital samples, etc.). The sampling was exhaustive and non-probability. Only the first unique isolate per patient was included to avoid duplication bias.

Sample Size Determination

The sample size was calculated using Cochran’s formula:

$N=\frac{z^2.P.(1-P)}{e^2}$

N: sample size

Z-score: Z corresponding to the desired confidence level (e.g., 1.96 for 95%)

P: estimated proportion of the population possessing the characteristic of interest (if unknown, 0.5 is used to ensure maximum sample size)

e: margin of error or desired precision (e.g., 5%, or 0.05)

Assuming a 95% confidence level, a 5% margin of error, and an unknown population proportion, the sample size was computed as follows:

$N=\frac{{(1,96)}^2\times 0,5\times (1-0,5)}{{(0,05)}^2}=384,16$

Therefore, the final sample size of end users to be surveyed was rounded up to 385 individuals.

Inclusion

The MDR bacterial strains included in this study were extended-spectrum beta-lactamase-secreting Enterobacteriaceae (ESBL-E), carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Pseudomonas aeruginosa (CRPA), isolated from various pathological products during the study period, regardless of their origin. Duplicate strains or strains not meeting our inclusion criteria and unusable data were excluded from this study.

Study variables

The dependent variable was the in vitro ceftazidime/avibactam (CZA) test performed on multidrug-resistant bacteria. The independent variables were the sociodemographic characteristics (sex, age, origin) and the clinical and bacteriological characteristics of the patient (type of sample, isolated species, and resistance type and phenotype of the identified multidrug-resistant bacteria).

Isolation and identification of MDR bacteria isolates in the laboratory

Non-duplicate, clinically significant MDR bacteria isolates were collected from patients. Clinical isolates were cultured on appropriate agar plates and incubated at 37°C for 24 hours. Then, isolated colonies were examined as follows: naked-eye examination of colonial morphology, microscopic examination of Gram-stained films, and further identification of the isolates by routine bacteriological methods using the oxidase test, Analytical Profile Index (API) 20E, and API 20 NE (bioMérieux, Marcy l’Étoile, France).

The agar disk diffusion method (Kirby-Bauer) was used to determine the phenotypes of multidrug-resistant bacteria, following the latest recommendations from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2024. While broth microdilution is the reference method for CZA testing, the Kirby-Bauer disk diffusion method was employed due to its practicality and cost-effectiveness in our routine laboratory setting, and it provides a reliable screening tool. The double-disc synergy test was used to detect ESBL-producing Enterobacteriaceae. The ceftazidime (10μg) disc was deposited 30 mm from the amoxicillin + clavulanic (10μg/20μg). A positive outcome is noted if a disc containing clavulanic acid crosses the ceftazidime (10μg) disc inhibitory zone: Meropenem (10μg) and imipenem (10μg) in detecting carbapenem-resistant strains.

Bacteria isolates are described as MDR if non-susceptible to ≥ 1 agent in ≥ 3 antimicrobial categories [16].

In vitro sensitivity test to ceftazidime/avibactam

Isolate identification and resistance phenotypes were first confirmed. A 0.5 McFarland standard suspension of each strain was cultured onto the Mueller–Hinton agar according to the EUCAST guidelines (2024) [16]. A Ceftazidime/Avibactam (14 μg) disk was placed in the center of the plate using sterile forceps. After incubating the plate at 37°C overnight, the zone of inhibition around the disk was measured. The zone of inhibition was interpreted as resistant, intermediate, and sensitive according to EUCAST guidelines 2024 [16].

Quality control

The reference strain Escherichia coli ATCC 25922 was used for quality control of antibiotic discs. This strain was tested weekly to ensure inter-laboratory reproducibility and accuracy.

Statistical analysis

SPSS 26.0 (Statistical Package for the Social Sciences) was used for the statistical analysis. All frequencies, including susceptibility and resistance to ceftazidime/avibactam, were provided as a percentage for each material type, microorganism type, ESBL-E and carbapenem-resistance, and drug resistance type. Chi-square tests were used to compare resistance rates across bacterial groups, with p-values < 0.05 considered statistically significant. Logistic regression was performed to determine the associations between dependent and independent variables. The adjusted odds ratio and 95% confidence interval were used to assess the strength of the association.

Ethical consideration

The study was conducted in compliance with the international ethical guidelines for biomedical research involving human subjects. This study was approved by the National Commission for Ethics for Research in the Health Sciences of Niger (date 03.04.2025/no 16/2025/CNERS). All the information collected from the patients was available for clinical use and research purposes. Privacy rules were maintained, and confidentiality was observed at all levels of data handling.

Results

A total of 306 non-duplicate bacteria were isolated from various biological samples collected from different patients.

Baseline characteristics of patients

The median age of patients was 49 (IQR: 20-68 years). The patients ranged in age from 3 days to 97 years, and a greater percentage were≥ 40 years old (61.1%). Most of our study population was male (sex ratio: 2.73). Gram-negative bacilli were isolated more frequently in the urine samples (85.9%) (Table 1).

Table 1.

Baseline characteristics of included patients

Parameters	Categories	Frequency	%
Sex	Male	224	73.2
	Female	82	26.8
	< 40	119	38.9
Age groups (years)	≥ 40	187	61.1
	Urine†	263	85.9
Type of samples	Pus	40	13.1
	Others††	3	1.0
Provenance	Inpatients	211	68.9
	Outpatients	95	31.1
	Medical	162	76.8
Departments	Emergencies and ICU†††	39	18.5
	Surgical	10	4.7

†Urine: issues by miction and on probe; ††Others: blood culture (n=1), Stool culture (n=1), Cerebrospinal fluid culture (n=1); †††ICU: intensive care unit

Frequency of MDR bacteria isolates

Of the 306 strains isolated, 286 (93.5%) were MDR. Among the MDR isolated, ESBL-E (n=198) and CRE (n=47) were the most represented (Table 2).

Table 2.

MDR bacteria isolates collected.

Type	Phenotypes	Frequency	%
	ESBL-E	198	64.7
MDR (n=286)	CRAB	14	4.6
	CRE	47	15.4
	CRPA	27	8.8
	CSAB	6	2.0
No MDR (n=20)	CSE	11	3.6
	CSPA	3	1.0
	Total	306	100.0

CSAB: Carbapenem-susceptible Acinetobacter baumannii; CSE: Carbapenem-susceptible Enterobacteriaceae; CSPA: Carbapenem-susceptible Pseudomonas aeruginosa.

Antibiotic resistance patterns of MDR isolates

Among the antibiotics tested, resistance to cotrimoxazole was most prevalent across the different phenotypes of the MDR bacteria collected. CRE and CRPA were the most resistant to cotrimoxazole, with 100% frequency, followed by ESBL, with 94.4%. Gentamicin and Nitrofurantoin were inactive in CRAB and CRPA (Figure 1).

Figure 1.

Resistance profile of MDR bacteria to the antibiotics

ESBL-E: Extended-spectrum β-lactamases producing Enterobacterales; CRE: Carbapenem-resistant Enterobacteriaceae; CRAB: Carbapenem-resistant Acinetobacter baumannii; CRPA: Carbapenem-resistant Pseudomonas aeruginosa

Frequency of resistance to ceftazidime / avibactam

Overall, 23.4% (n=67) of the isolated MDR bacteria exhibited resistance to ceftazidime/avibactam (Figure 2a). CRAB (100%) had the highest resistance to ceftazidime/avibactam, followed by CRE (61.7%), CRPA (33.3%), and ESBL-E (7.6%) (Figure 2b). A chi-square test confirmed a statistically significant difference in resistance rates among these phenotypes (p < 0.001).

Figure 2.

Resistance to ceftazidime/avibactam. (a): Frequency of resistance to ceftazidime/avibactam; (b): ceftazidime/avibactam in vitro activity according to the phenotype of the isolates.

ESBL-E: Extended-spectrum beta-lactamases producing Enterobacterales; CRE: Carbapenem-resistant Enterobacteriaceae; CRAB: Carbapenem-resistant Acinetobacter baumannii; CRPA: Carbapenem-resistant Pseudomonas aeruginosa.

Associated factors with resistance to ceftazidime / avibactam

In univariate (Table 3) and multivariate analysis (Figure 2), any of the following variables, groups of MDR, phenotypes of MDR, fluoroquinolones, aminoglycosides, and carbapenems, were significantly associated with resistance to ceftazidime/avibactam.

Table 3.

Univariate analysis of factors associated with resistance to ceftazidime/avibactam

		Ceftazidime/Avibactam		Odds ratio	CI 95%	p-value
Variables		Active n (%)	Non-active n (%)
	Female	59 (20.6)	16 (5.6)	1	-	-
Sex	Male	160 (55.9)	51 (17.8)	1.26	0.72-2.26	0.416
	< 40	91 (31.8)	22 (7.7)	1	-	-
Age (years)	≥ 40	128 (44.8)	45 (15.7)	1.27	0.68-2.37	0.448
	Others	1 (0.3)	2 (0.7)	1	-	-
Samples	Pus	21 (7.3)	11 (3.8)	1.50	0.2711.63 -	0.657
	Urine	197 (68.9)	54 (18.9)	1.27	0.27-9.02	0.776
	Outpatient	64 (22.4)	18 (6.3)	1	-	-
Provenance	Impatient	155 (54.2)	49 (17.1)	0.78	0.47-1.28	0.329
	Emergencies	34 (16.7)	8 (3.9)	1	-	-
Departments	Medical	114 (55.9)	40 (19.6)	1.56	0.69-4.43	0.250
	Surgical	7 (3.4)	1 (0.5)	1.72	0.70-3.70	0.287
	Enterobacteria	201 (70.3%)	44 (15.4%)	1	-	-
Groups of MDR	NFGNB	18 (6.3%)	23 (8.0%)	5.76	2.8812.09 -	0.001 <
	ESBL-E	183 (64.0)	15 (5.2)	1	-	-
	CRE	18 (6.3)	29 (10.1)	16.13	7.86-	<
Phenotypes					35.04	0.001
	PARC	18 (6.3)	9 (3.1)	13.10	5.4234.48 -	0.001 <
	Active	44 (15.4)	11 (3.8)	1	-	-
Fluoroquinolones	Non-active	175 (61.2)	56 (19.6)	2.29	1.05-5.53	0.048
	Active	62 (21.7)	14 (4.9)	1	-	-
Aminoglycosides	Non-active	157 (54.9)	53 (18.5)	1.94	1.06-3.72	0.038
	Active	184 (64.3)	15 (5.2)	1	-	-
Carbapenems	Non-active	35 (12.2)	52 (18.2)	19.00	9.9638.27 -	0.001 <

CI 95%: confidence interval at 95%; NFGNB: Non-fermenting Gram-negative bacilli; ESBL-E: Extended-spectrum beta-lactamase Enterobacteriaceae; CRE: Carbapenem-resistant Enterobacteriaceae;

CRPA: Carbapenem-resistant Pseudomonas aeruginosa.

Figure 3.

Multivariate analysis of factors associated with resistance to ceftazidime/avibactam.

CI95%: Confidence interval at 95%; ESBL-E: Extended spectrum beta-lactamase Enterobacteriaceae;

CRE: Carbapenem-resistant Enterobacteriaceae; CRPA: Carbapenem-resistant Pseudomonas aeruginosa

Discussion

This study provides one of the first detailed assessments of the in vitro activity of ceftazidime/avibactam against multidrug-resistant Gram-negative bacilli in Niger. The key finding is the dramatic divergence in its efficacy based on the resistance phenotype: preserved activity against the majority of Extended-spectrum β-lactamase-producing Enterobacterales, but near-ubiquitous resistance in carbapenem-resistant strains, particularly Acinetobacter baumannii.

Our study reveals an overall CZA resistance rate of 23.4% among MDR isolates. This figure actually masks two opposing realities. CZA demonstrates excellent activity against ESBL-E, with a susceptibility rate of 92.4% (183/198). This result aligns with the pharmacological profile of the molecule, as avibactam effectively inhibits ESBLs and AmpC cephalosporinases. This performance is comparable to the INFORM Global Surveillance Study from 2012 to 2014, which reported a susceptibility rate of 99.5% for ESBL-E [17], and confirms the role of CZA as a first-line option for treating severe ESBL-E infections in our setting [18].

However, we observed alarming resistance in non-fermenting bacilli and carbapenem-resistant Enterobacterales. Resistance rates of 100% (14/14) for CRAB, 61.7% (29/47) for CRE, and 33.3% (9/27) for carbapenem-resistant Pseudomonas aeruginosa. This hierarchy is consistent with the literature, but the magnitude of the rates is concerning. A study conducted in Italy reported a 100% rate of carbapenem resistance in CRE-producing organisms due to the NDM gene.[19] a rate already considered high but higher than what we observe here (61.7%). On the other hand, a study conducted in Africa reported a resistance rate of 15% among CRE producing the OXA-48 type resistance gene, a frequency lower than that observed in our study [20].

Multivariate analysis identified a strong and independent association between carbapenem resistance and CZA resistance (OR=19.00; 95% CI: 9.96-38.27). This correlation is a strong indicator of the underlying resistance mechanisms.

In CRE: CZA resistance in CRE is primarily attributable to the production of Metallo-Beta-Lactamase (MBL)-type carbapenemases, such as NDM, VIM, or IMP. Indeed, avibactam is ineffective at inhibiting these enzymes. The high prevalence of MBLs in Africa and Asia is well-documented [21,22]. Our resistance rate of 61.7% strongly suggests that MBLs predominate among circulating CRE in Niger. Other, rarer mechanisms can contribute, such as the combined production of a KPC-type carbapenemase and porin mutations, which reduce permeability to the molecule [23,24].

In CRAB, the 100% resistance to CZA was expected. Acinetobacter baumannii has intrinsic resistance to CZA, as reported in the literature, mainly due to the presence of a native AmpC cephalosporinase, ADC, which avibactam inhibits poorly. In carbapenem-resistant strains, this weak activity is overridden by the acquisition of OXA-23, OXA-58, or MBL carbapenemases, rendering the molecule completely inactive [25].

In CRPA, the 33.3% resistance rate is significant. It can be explained by several mechanisms: production of MBLs, mutations affecting outer membrane permeability (OprD porin) coupled with overexpression of efflux pumps, or specific mutations in the PDC cephalosporinase that reduce affinity for avibactam [26].

Limitations

This study, conducted at the National Reference Laboratory for Antimicrobial Resistance in Niger, demonstrated strong in vitro activity of ceftazidime/avibactam against multidrug-resistant Gram-negative bacilli carrying ESBL-E, CRE, and CRPA, but limited efficacy against CRAB isolates. To the best of our knowledge, this is the first study on the in vitro activity of ceftazidime/avibactam on multidrug-resistant bacteria in Niger.

However, this study has several limitations. Due to limited resources, this article focused on the phenotype of strains and did not detect genes for resistance to Ceftazidime/avibactam in multidrug-resistant bacterial strains. Additionally, the association between Ceftazidime/avibactam treatment and patient outcomes in patients with multidrug-resistant bacterial infections could not be analyzed due to insufficient data. Furthermore, this was a single-center study conducted at the national reference laboratory, and the isolates may not be fully representative of the resistance patterns across all healthcare facilities in Niger. The lack of molecular characterization limits our ability to identify the underlying resistance mechanisms definitively.

Conclusion

In this study, the susceptibility of multidrug-resistant Gram-negative bacilli to Ceftazidime/Avibactam is relatively high. Ceftazidime/avibactam is still a viable option for treating infections caused by ESBL-E when no other alternatives are available. Additional research is required to explore alternative strategies for addressing CRAB, CRE, and CRPA. These findings highlight the importance of using ceftazidime/avibactam carefully to maintain its effectiveness. To ensure proper monitoring of its efficacy, ceftazidime/avibactam should be included in routine laboratory antibiogram susceptibility testing to prevent premature and inappropriate widespread use. This study establishes a high in vitro efficacy of CZA against ESBL-E in Niger, supporting its introduction into routine susceptibility testing to guide targeted therapy. However, the high resistance observed in CRAB and CRE necessitates caution and underscores that CZA is not a panacea. To preserve its utility, we recommend: 1) the immediate integration of CZA into routine antibiograms, 2) investment in molecular surveillance to track resistance mechanisms, and 3) the development of local treatment guidelines that reflect these distinct susceptibility profiles.