# Susceptibility of some Enterobacteriaceae isolated from 4 different aquatic environments in DR Congo (Central Africa), to Amoxicillin/Clavulanic acid and some 3rd generation Cephalosporins

Mwanzo Wavindu, Edith Mouafo Tamnou, Ildephonse Soly Kamwira, Ngome M Rosanne, Paul Alain Nana, Geneviève Bricheux, Maximilienne Ascension

Nyegue, Télesphore Sime-Ngando, Moïse Nola

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Mwanzo Wavindu, Edith Mouafo Tamnou, Ildephonse Soly Kamwira, Ngome M Rosanne, Paul Alain Nana, et al.. Susceptibility of some Enterobacteriaceae isolated from 4 different aquatic environments in DR Congo (Central Africa), to Amoxicillin/Clavulanic acid and some 3rd generation Cephalosporins. GSC Biological and Pharmaceutical Sciences, 2024, 26 (1), pp.178 - 190. 10.30574/gscbps.2024.26.1.0002 . hal-04769704

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# Susceptibility of some Enterobacteriaceae isolated from 4 different aquatic environments in DR Congo (Central Africa), to Amoxicillin/Clavulanic acid and some 3rd generation Cephalosporins

Mwanzo Wavindu 1, 2, 3, Edith B. Mouafo Tamnou 1, Ildephonse Soly Kamwira 2, Rosanne M. Ngome 4, Paul Alain Nana 5, Geneviève Bricheux 6, Maximilienne A. Nyegue 3, Télesphore Sime-Ngando 7 and Moïse Nola 1,*

GSC Biological and Pharmaceutical Sciences, 2024, 26(01), 178-190

Publication history: Received on 26 November 2023; revised on 06 January 2024; accepted on 09 January 2024

Article DOI: https://doi.org/10.30574/gscbps.2024.26.1.0002

## Abstract

Little is known about the variation of the antibiotic susceptibility of different bacterial strains of the same cells species, isolated from different aquatic environments. The present study aims to evaluate the susceptibility towards some 3rd generation cephalosporins (Ceftriaxon, Ceftazidim and Cefotaxim) and Amoxicillin/Clavulanic acid, of Enterobacteriaceae strains isolated from groundwater, stream, hospital wastewater and slaughterhouse wastewater. Enterobacteria species were isolated on Mac Conkey agar, then identified using enzymatic and MALDI-TOF MS system. The antimicrobial susceptibility was carried out using the disk diffusion method. The antibiotic minimum inhibitory concentrations (MICs) were carried using the VITEK®2 system. Bacterial species mostly identified were *Klebsiella pneumoniae*, *Escherichia coli*, *Salmonella typhi* and *Ewingella americana*. The antibiotic inhibition diameters and the MICs varied depending on the antibiotics, bacterial species and type of aquatic environment hosting the microorganism. Relatively lower MICs were recorded with Cefotaxim against different bacteria in slaughterhouse wastewater, in river water and in well water. Antibiotic resistance was noted with all strains from hospital wastewater. Significant differences (P<0.05) amongst antibiotic inhibition diameter was noted for *K. pneumoniae* and *S. typhi* in most cases. The relative variations in the action mechanisms amongst antibiotics and the intrinsic defense potential of each bacterial strain, as well as the potential influence of the physicochemical properties of each water medium, could partly be at the origin of the relative differences observed at the phenotypic level. It seems necessary to explore the diversity, similarities and differences amongst antibiotic resistance genes in these different types of aquatic biotopes.

**Keywords:** Aquatic environments; Enterobacteriaceae; 3rd generation Cephalosporins and Amoxicillin/Clavulanic acid; Antibiotic inhibition diameters and MICs; Antibiotic susceptibility variation

<footnote n="1">¹ Laboratory of Hydrobiology and Environment, Faculty of Sciences, University of Yaounde I, Cameroon.</footnote>

<footnote n="2">² Central Research Laboratory, Faculty of Pharmaceutical Sciences, Catholic University of Graben, DR Congo.</footnote>

<footnote n="3">³ Laboratory of Medical Microbiology, Faculty of Sciences, University of Yaounde I, Cameroon.</footnote>

<footnote n="4">⁴ Service de Bactériologie-Parasitologie-Mycologie, Centre Pasteur, BP 1274 Yaounde, Cameroun.</footnote>

<footnote n="5">⁵ Department of Oceanography and Limnology, Institute of Fisheries and Aquatic Sciences, University of Douala, Cameroon.</footnote>

<footnote n="6">⁶ Laboratoire "Microorganismes: Génome et Environnement" (LMGE), UMR CNRS 6023, Université Clermont Auvergne, Campus Universitaire des Cézeaux, 1 Impasse Amélie Murat, TSA 60026 - CS 60026, 63178 Aubière Cedex, France.</footnote>

<footnote n="7">⁷ Laboratoire « Microorganismes: Génome et Environnement » (LMGE), UMR CNRS 6023 et Laboratoire Magmas et Volcans (LMV), UMR IRD 163. Campus Universitaire des Cézeaux, TSA 60026 - CS 60026, 63178 Aubière Cedex, France.</footnote>
# 1. Introduction

Cephalosporins are a large group of antibiotics derived from the mold *Acremonium* (previously called *Cephalosporium*). Because of their effectiveness against a wide range of Gram negative bacteria, they are often called broad-spectrum antibiotics [1, 2]. They bind to and block the activity of enzymes responsible for making peptidoglycan, an important component of the bacterial cell wall. Drugs known as cephalosporins included ceftriaxon, cefdinir, cefixim, cefpodoxim, cefditoren, ceftibuten, cefdinir, ceftazidim, cefotaxim, cefoperazone, ceftizoxim, among others [2]. 3rd generation Cephalosporins included Cefixim, Cefpodoxim, proxetil, Cefotaxim, Ceftazidim and Ceftriaxon. They diffuse well in tissues. Cefotaxime, ceftazidime and ceftriaxone have the particularity of crossing the blood-brain barrier, and are therefore effective in the treatment of meningitis, unlike other 3rd generation Cephalosporins [3].

Amoxicillin works by binding to penicillin-binding proteins and inhibiting peptidoglycan synthesis, which interrupts the construction of the cell wall and ultimately leads to the destruction, or lysis, of the bacteria. With the addition of clavulanic acid, the spectrum Amoxicillin-clavulanate is increased to include all beta-lactamase-producing strains and are often enterobacteria [4].

Enterobacteriaceae is a family of Gram-negative rod-shaped (bacilli) bacteria in the order Enterobacterales of the class Gammaproteobacteria in the phylum Pseudomonadota, domain of Bacteria [5]. Recently based on combined results of phenotypic properties, DNA sequences and DNA homology, phylogenetic findings, and 16S rRNA gene sequence study results, 68 genera with a total of 355 species have been proposed [5, 6]. They are cosmopolitan bacteria. They are found in soil, decaying vegetation and organic matters, water sources, sewage, and other environmental aspects. Most of them are mesophilic, hence they are not found in extremely cold and extremely hot environments. They are found scarcely in high salinity, xerophilic (extremely dry), and extreme pH areas [5].

They are commonly known as Gram-negative, rod-shaped (usually short bacilli, about 1-5 µm), facultatively anaerobic, non-sporing, mostly motile with peritrichous flagella (most species in the genera *Klebsiella* and *Shigella* are non-motile), catalase positive, oxidase negative, both lactose fermenter and non-fermenter, usually acid-producing and nitrate-reducing. In addition, the outer membrane (O), flagella (H), and capsule (K) are antigens in most species [5, 7]. In humans, many new species have been described, some associated with specific disease processes. Some established species are now observed in new infectious disease settings and syndromes [6].

Many bacterial species have been reported in groundwater with different antibiotic susceptibilites [8]. During bacterial infiltration through soil layers, different microbiogeochemical reactions and interactions can occur [9, 10]. In groundwater as in river water, different interactions can lead to potential genetic mutations of microorganisms [11]. This could influence the relevant bacterial susceptibility against antibiotics such as Cephalosporins or Amoxicillin/clavulanic acid, and sometimes make it difficult to fight against an infection whose germs are of different aquatic origins. It therefore seems necessary to evaluate, both in rural or in urbanized areas, the variation in susceptibility to Amoxicillin/Clavulanic acid and 3rd generation Cephalosporins (which are the most used to fight infections) of enterobacteria present at the same time in several aquatic environments of different properties.

Waters encountered on continents are often groundwater, streams and wastewater which may be hospital or come from slaughterhouses. In general, they are of different biotic and abiotic properties. Little is known about the susceptibility to the same antibiotic, of different bacterial strains of the same cells species, isolated from different aquatic environments. The present study aims to evaluate the susceptibility towards some 3rd generation Cephalosporins and Amoxicillin/Clavulanic acid, cells strains of some enterobacteria species isolated simultaneously from groundwater, stream, hospital wastewater and slaughterhouse wastewater in an urban area in the Democratic Republic of Congo (DR Congo) (Central Africa).

# 2. Materials and methods

## 2.1. Study area and sampling sites

The study was carried out in the city of Butembo, in the east of the DR Congo. Four different types of aquatic environments were choose. They included hospital wastewater (HW), slaughterhouse wastewater (SW), river water (RW) and wells (W). Sampling sites are of different geographic coordinates. The latitudes were 0°7′17.346″ for SW, 0°7′24.8016″ for HW, 0°5′38.06988″ for RW and 0°6′35.3366″ for W. The longitudes were 29°17′10.39812″ for SW, 29°15′48.76776″ for HW, 29°19′32.7576″ for RW and 29°18′42.93288″ for W. Altitudes were 1724m for SW, 1761m for HW, 1807m for RW and 1734m for W. The total depth of this well point was 6m.
## 2.2. Water samplings

The study was carried out during the period from August 2022 to February 2023. This period covers the two main seasons of the locality, which are the rainy season (August-December) and the dry season (January and February). Each water point was sampled once a month. Water samples were taken in sterile 500 mL glass bottles for microbiological analyses, and clean 1000 mL polyethylene bottles for physicochemical analyses. All the samples were then placed in an isothermal container and immediately brought back to the laboratory for analysis.

## 2.3. Bacteriological analyses

### 2.3.1. Isolation and identification of Enterobacteriaceae

After homogenization of the water samples, followed or not by decimal dilutions, the isolation of the Enterobacteriaceae was done by spreading on the surface on Mac Conkey agar (Titan Biotech LTD: W/CV, NaCl, 0,15% Bile salts and 1% Lactose) poured into petri dishes and then incubated at 37 °C for 18 to 24 hours [12].

The macroscopic observation consisted of examining the cultural characters of the bacteria expressed in colonies. These include the color, size, contours and surface configuration of the colony. After purification of the strains, their identification was carried out first by the enzymatic method [12], at Central Research Laboratory, Faculty of Pharmaceutical Sciences, Catholic University of Graben, DR Congo, then using the Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [13, 14], at the Pasteur Center of Yaounde (Cameroon). The mass-to-charge (m/z) ratios are electrodynamic measurements of how quickly charged ions from the clinical sample material move through the TOF tube and reach a detector.

From of 18 to 24 hours culture, selected colony was applied onto MALDI test plant. Samples were then overlaid with matrix and dried. The plate was subsequently loaded into the MALDI-TOF MS instrument: the sample was bombarded by the laser. This bombardment resulted in the sublimation and ionization of both the sample and matrix. These generated ions were separated based on their mass-to-charge ratio via a TOF tube, and a spectral representation of these ions was generated and analyzed by the MS software, generating an MS profile. This profile was subsequently compared to a database of reference MS spectra and matched to either identical or the most related spectra contained in the database, generating an identification for bacteria and analyzed by software associated with the respective system, allowing rapid identification of the microorganism [13-15].

### 2.3.2. Nutrient media and culture conditions

The medium used was Müller Hinton agar (Biorad) poured into Petri dishes. The thickness of the agar was approximately 4 mm. The surface of the agar was dried before use [16]. From an 18-24 h culture on non-selective agar medium (Plate Count Agar), a bacterial suspension in saline solution (0.9% NaCl) with a turbidity equivalent to that of the standard 0.5 of the range of McFarland was carried out, which corresponds to a bacterial density of approximately $1 \times 10^8$ CFU/100 mL The inoculum was then diluted 1/10 ($1 \times 10^7$ CFU/100 mL) before inoculation [16].

The agar was inoculated with the bacterial inoculum by the swab method. The entire surface of the agar was swabbed in three directions. The antibiotic discs were placed on the surface of the inoculated and dried agar. The gap between the discs was 3 cm in order to avoid overlapping of the inhibition diameters. The Petri dishes were then incubated within 15 min following the depositing of the discs, at 37 °C aerobically for 24 h [16].

### 2.3.3. Antibiotic susceptibility assay

The antimicrobial susceptibility tests were carried out using the disk diffusion method according to the recommendations of the FMS-EUCAST [16]. The antibiotic molecules were chosen in depending on the uses of the population but also on their availability in the laboratory. Those considered were Ceftriaxon, Ceftazidim, Cefotaxim, and Amoxicillin+Clavulanic acid

The inhibition diameters (ID) were measured using the caliper and the results were scored as resistant, sensitive or intermediate according to CA-SFM recommendations [16, 17]. Ceftriaxon, Ceftazidim and Cefotaxim were scored sensitive when ID≥21mm, intermediate when 20mm≤ID≥15mm, and resistant when ID<15mm. Amoxicillin+Clavulanic acid was scored sensitive when ID≥21mm, intermediate when 20mm≤ID≥14mm, and resistant when ID<14mm.

The antibiotic minimum inhibitory concentrations (MICs) against each of the bacteria considered were carried out at the Pasteur Center of Yaounde (Cameroon) using the VITEK®2 technic [18]. VITEK®2 Compact Systems utilize automated growth-based detection using attenuation of light measured by an optical scanner. The optics used in the
systems use visible light to directly measure organism growth. Transmittance optics are based on an initial light reading of a well before significant growth has begun. Periodic light transmittance samplings of the same well measure organism growth by how much light is prevented from going through the well. The VITEK®2 System monitors the growth of each well in the card over a defined period of time. An interpretive call is made between 4 and 16 hours for a “rapid” read but may be extended to 18 hours in some instances. At the completion of the incubation cycle, a report is generated that contains the MIC value along with the interpretive category result for each antibiotic on the card.

From 18h-24h grown colonies a calibrated bacterial suspension in sodium chloride solution, was adjusted to an optical density of 0.5-0.55 McF. A selection of drugs relevant to sepsis management in AST-N233+XN05. Results were automatically reported by Automate Expert System software version 8.01 running with 2018 EUCAST/CA-SFM breakpoints. MICs and interpreted categories expertise by VITEK®2 AES (Advanced Expert System) from D0 workflow were compared to those from D1 workflow [18].

## 2.4. Physicochemical analyses

The parameters considered included water pH, electrical conductivity, turbidity, nitrite and nitrate concentrations. Some of them (water pH and electrical conductivity) were measured in the field using a multi-parameter HACH/POCKET PRO+ and the others were measured in the laboratory using a spectrophotometer CECIL CE1011. This was done according to the recommended techniques [19].

## 2.5. Data analysis

The values of the antibiotic inhibition diameters were illustrated by histograms plotted using Excel 2010 software. The values of the antibiotic inhibition diameters were illustrated by histograms plotted using Excel 2010 software. Using the Kruskal-Wallis test, the comparison amongst antibiotic inhibition diameters has been carried out using SPSS version 20.0 software (a value of P<0.05 was assumed to be significant).

# 3. Results

## 3.1. Spatio-temporal variation of the presence of bacterial species in the aquatic environments considered

A total of 4 bacteria species identified were most often isolated. These are *Klebsiella pneumoniae*, *Escherichia coli*, *Salmonella typhi* and *Ewingella americana* (Figure 1).

<figure><img src="image_3.png" /><figcaption>**Figure 1** Presence or absence of each bacterial strains considered in each of the aquatic environment of water during the sampling period</figcaption></figure>
The frequency of presence or absence varied from one biotope to another. For example, in slaughterhouse wastewater, none of these species was identified during the month of November 2022. The same is true of hospital wastewater during the month of August 2022, as well as in January 2023 in river waters. In the well water, at least one of the bacterial species considered was identified in each month of the study period (Figure 1). In the 4 different aquatic biotopes sampled, *S. typhi* and *K. pneumoniae* were most often isolated. Additionally, *E. coli* has been isolated several times from well water and hospital wastewater (Figure 1).

## 3.2. Spatial variation of the antibiotics inhibition diameters with respect to each bacteria species and aquatic environment considered

The inhibition diameters varied from one antibiotic to another, from one biotope to another and from one bacterium to another. The inhibition diameters recorded with Amoxicillin+Clavulanic acid were relatively low (6-12mm) in the most of cases, with the exception for *K. pneumoniae* (20mm) isolated from slaughterhouse wastewater and for *S. typhi* (24mm) isolated from river waters. For strains isolated from hospital wastewater, the inhibition diameters recorded varied from 6-12mm; an exception was observed for *K. pneumoniae* (Ceftriaxone: 18mm and Ceftazime: 16mm) (Figure 2).

<figure><img src="image_4.png" /><figcaption>**Figure 2** Mean values of the antibiotics inhibition diameters during the sampling period with respect to each bacteria strains and aquatic biotope considered (hospital wastewater (HW), slaughterhouse wastewater (SW), river water (RW) and wells (W))</figcaption></figure>

## 3.3. Spatial variation of the antibiotics MIC with respect to each bacteria species and aquatic environment considered

The MICs varied depending on the antibiotics, from one bacterium to another on the one hand, and depending on the aquatic biotope hosting the germ on the other hand. Most of the high MICs (>16 and >32 µg/ml) were recorded in hospital wastewater for the four drugs tested. The MICs of Amoxicillin+clavulanic acid were relatively higher (>16 µg/ml) against the different bacteria in all the biotopes explored. Relatively low MICs (≤ 1 µg/ml) were recorded with Cefotaxime against different bacteria in slaughterhouse wastewater, in river water and in well water. The same is true for Ceftazidim in wastewater from the slaughterhouse and wells (Table 1).
<table><caption>**Table 1** Ranges of MIC values depending on the bacteria and the aquatic biotope sampled</caption><thead><tr><th colspan="2">Antibiotic used and aquatic biotope sampled</th><th colspan="4">MIC value</th></tr><tr><th>Antibiotic</th><th>Aquatic environment</th><th>Klebsiella pneumoniae</th><th>Salmonella typhi</th><th>Escherichia coli</th><th>Ewingella americana</th></tr></thead><tbody><tr><td rowspan="4">Ceftriaxon</td><td>SW</td><td>≤ 4</td><td>≤ 4</td><td>≤ 4</td><td>≤ 4</td></tr><tr><td>HW</td><td>8</td><td>>32</td><td>>32</td><td>>32</td></tr><tr><td>RW</td><td>≤ 4</td><td>≤ 4</td><td>≤ 4</td><td>8</td></tr><tr><td>W</td><td>≤ 4</td><td>≤ 4</td><td>≤ 4</td><td>≤ 4</td></tr><tr><td rowspan="4">Cefotaxim</td><td>SW</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td></tr><tr><td>HW</td><td>8</td><td>>32</td><td>>32</td><td>>32</td></tr><tr><td>RW</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td></tr><tr><td>W</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td></tr><tr><td rowspan="4">Ceftazidim</td><td>SW</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td></tr><tr><td>HW</td><td>>32</td><td>>32</td><td>>32</td><td>>32</td></tr><tr><td>RW</td><td>16</td><td>16</td><td>16</td><td>16</td></tr><tr><td>W</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td><td>≤ 1</td></tr><tr><td rowspan="4">Amoxicillin+Clavulanic acid</td><td>SW</td><td>2</td><td>>16</td><td>10</td><td>>16</td></tr><tr><td>HW</td><td>>16</td><td>>16</td><td>>16</td><td>>16</td></tr><tr><td>RW</td><td>>16</td><td>2</td><td>>16</td><td>>16</td></tr><tr><td>W</td><td>>16</td><td>>16</td><td>>16</td><td>>16</td></tr></tbody></table>

**3.4. Antibiotic scored susceptibility**

From the mean antibiotic inhibition diameters indicated above, antibiotic susceptibility has been assessed with respect to bacteria considered and aquatic biotope sampled. Results are shown in Table 2. Resistance to the four antibiotic molecules is noted in all bacterial species in hospital wastewater. The four bacterial species are resistant to Amoxicillin+Clavulanic acid in the different biotopes explored. An exception was noted for *K. pneumoniae* in slaughterhouse wastewater and *S. typhi* in river water. Susceptibility of all four bacterial species to Ceftriaxone, Cefotaxime and Ceftazime was observed in river water, well water and slaughterhouse wastewater (Table 2).

<table><caption>**Table 2** Antibiotic susceptibility with respect to bacteria considered and aquatic biotope sampled</caption><thead><tr><th colspan="2">Antibiotics used and aquatic biotope sampled</th><th colspan="4">Bacteria considered and the antibiotic susceptibility</th></tr><tr><th>Antibiotics</th><th>Aquatic biotope</th><th>Klebsiella pneumoniae</th><th>Salmonella typhi</th><th>Escherichia coli</th><th>Ewingella americana</th></tr></thead><tbody><tr><td rowspan="4">Ceftriaxon</td><td>SW</td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td>HW</td><td>I</td><td>R</td><td>R</td><td>R</td></tr><tr><td>RW</td><td>S</td><td>S</td><td>S</td><td>I</td></tr><tr><td>W</td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td rowspan="2">Cefotaxim</td><td>SW</td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td>HW</td><td>I</td><td>R</td><td>R</td><td>R</td></tr></tbody></table>
<table><tbody><tr><td></td><td>RW</td><td>S</td><td>S</td><td>S</td><td>I</td></tr><tr><td></td><td>W</td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td rowspan="4">Ceftazidim</td><td>SW</td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td>HW</td><td>R</td><td>R</td><td>R</td><td>R</td></tr><tr><td>RW</td><td>S</td><td>S</td><td>S</td><td>I</td></tr><tr><td>W</td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td rowspan="4">Amoxicillin+Clavulanic acid</td><td>SW</td><td>S</td><td>R</td><td>I</td><td>R</td></tr><tr><td>HW</td><td>R</td><td>R</td><td>R</td><td>R</td></tr><tr><td>RW</td><td>R</td><td>S</td><td>R</td><td>R</td></tr><tr><td>W</td><td>R</td><td>R</td><td>R</td><td>R</td></tr></tbody></table>

### 3.5. Comparison amongst some considered parameters

Several comparisons of the antibiotics inhibition diameters have been carried out between the parameters considered. The first was made amongst antibiotics used for each bacteria and each aquatic environment sampled. Results are presented in Table 3. A significant difference in antibiotic inhibition diameter (P<0.05) is noted for *K. pneumoniae* in river water and in well water, for *S. typhi* in slaughterhouse wastewater, river and well water. A significant difference in antibiotic inhibition diameter (P<0.05) is also noted for *E. coli* in slaughterhouse wastewater, river water and well water. Finally, for *E. americana* a significant difference (P<0.05) is observed only in well water (Table 3).

<table><caption>**Table 3** "P" value of the comparison of inhibition diameters amongst antibiotics used for each bacteria and each aquatic biotope sampled</caption><thead><tr><th rowspan="2">Bacteria strains considered</th><th colspan="4">Aquatic biotopes sampled</th></tr><tr><th>Slaughterhouse wastewater</th><th>Hospital wastewater</th><th>River water</th><th>Wells</th></tr></thead><tbody><tr><td>Klebsiella pneumoniae</td><td>P=0.061</td><td>P=0.091</td><td>P=0.024*</td><td>P=0.025*</td></tr><tr><td>Salmonella typhi</td><td>P=0.027*</td><td>P=0.622</td><td>P=0.039*</td><td>P=0.024*</td></tr><tr><td>Escherichia coli</td><td>P=0.019*</td><td>P=0.971</td><td>P=0.024*</td><td>P=0.031*</td></tr><tr><td>Ewingelia americana</td><td>P=0.052</td><td>P=0.275</td><td>P=0.063</td><td>P=0.024*</td></tr></tbody></table>

The second comparison of inhibition diameters was carried out amongst bacteria considered for each antibiotics and each aquatic environment sampled. Results are presented in Table 4. A significant difference in inhibition diameter (P<0.05) is observed for cefotaxim in river water, for ceftazidim in slaughterhouse wastewater, and for amoxicillin+Clavulanic acid in water waste from the slaughterhouse and river water (Table 4).

<table><caption>**Table 4** "P" value of the comparison of inhibition diameters amongst bacteria considered for each antibiotics and each aquatic biotope sampled</caption><thead><tr><th rowspan="2">Antibiotic used</th><th colspan="4">Aquatic biotope sampled</th></tr><tr><th>Slaughterhouse wastewater</th><th>Hospital wastewater</th><th>River water</th><th>Wells</th></tr></thead><tbody><tr><td>Ceftriaxon</td><td>P=0.062</td><td>P=0.110</td><td>P=0.074</td><td>P=0.315</td></tr><tr><td>Cefotaxim</td><td>P=0.085</td><td>P=0.205</td><td>P=0.026*</td><td>P=0.099</td></tr><tr><td>Ceftazidim</td><td>P=0.036*</td><td>P=0.754</td><td>P=0.099</td><td>P=0.147</td></tr><tr><td>Amoxicillin+Clavulanic acid</td><td>P=0.019*</td><td>P=0.664</td><td>P=0.034*</td><td>P=0.189</td></tr></tbody></table>

<footnote>* : Significant difference (P<0.05)</footnote>

<footnote>* : Significant difference (P<0.05)</footnote>
The last comparison of the inhibition diameters carried out was amongst aquatic environment sampled for each bacteria and antibiotics considered. Results are presented in Table 5. A significant difference (P<0.05) is observed in *K. pneumoniae* for Ceftazidim and Amoxicillin+Clavulanic acid; in *E. coli* and *E. americana*, a significant difference is noted for Ceftriaxone, Cefotaxim and Ceftazidim. No significant difference was reported in *S. typhi* for the different antibiotics (Table 5).

<table><caption>**Table 5** "P" value of the comparison of inhibition diameters amongst aquatic biotope sampled for each bacteria and antibiotics considered</caption><thead><tr><th rowspan="2">Bacteria strains considered</th><th colspan="4">Antibiotic used</th></tr><tr><th>Ceftrixon</th><th>Cefotaxim</th><th>Ceftazidim</th><th>Amoxicillin+Clavulanic acid</th></tr></thead><tbody><tr><td>Klebsiella pneumoniae</td><td>P=0.078</td><td>P=0.075</td><td>P=0.021*</td><td>P=0.043*</td></tr><tr><td>Salmonella typhi</td><td>P=0.062</td><td>P=0.090</td><td>P=0.077</td><td>P=0.077</td></tr><tr><td>Escherichia coli</td><td>P=0.028*</td><td>P=0.017*</td><td>P=0.044*</td><td>P=0.058</td></tr><tr><td>Ewingelia americana</td><td>P=0.024*</td><td>P=0.023*</td><td>P=0.019*</td><td>P=0.532</td></tr></tbody></table>

### 3.6. Some physico-chemical parameters of the aquatic biotopes sampled

During the study period, electrical conductivity varied from 480 to 750 µS/cm in hospital wastewater, from 320 to 920 µS/cm in river water, from 340 to 1060 µS/cm in water waste from the slaughterhouse and 180 to 250 µS/cm in well water (Figure 3).

<figure><img src="image_5.png" /><figcaption>**Figure 3** Temporal variation of the considered physicochemical parameters in each of the sampled aquatic biotope</figcaption></figure>

The pH oscillated from 5.46 to 6.64 for the hospital wastewater, from 5.28 to 6.8 for the river water, from 6 to 7.4 for the slaughterhouse wastewater and from 6.07 to 6.5 for well water. The nitrite content varied from 0 to 0.3 mg/L in

<footnote>* : Significant difference (P<0.05)</footnote>
hospital wastewater, from 0 to 0.38 mg/L in river water, from 0 to 0.6 mg/L in slaughterhouse wastewater and 0 to 4.6 mg/L in river water. The concentration of nitrates varied from 0.05 to 8.86 mg/L in hospital wastewater, from 0.05 to 17.39 mg/L in river water, from 0.58 to 2, 01 mg/L in slaughterhouse wastewater and 0.5 to 5.86 mg/L in well water (Figure 3).

**4. Discussion**

Variations in the frequency of presence or absence of bacteria in the different aquatic biotopes sampled were noted. This frequency would depend on the one hand on the intrinsic properties of the bacteria allowing it to resist the different constraints of the environment. It would also depend on the chemical and nutritional properties of each biotope, allowing the survival of each bacteria to be maintained.

The present study showed that inhibition diameters of different antibiotics against germs from hospital wastewater are relatively lower in most cases. In addition, these strains are also resistant to the considered antibiotics in most cases. It is stated that antibiotics are used worldwide to treat and prevent bacterial infections in humans, animals and even plants. They are also widely used to stimulate the growth of animals to increase meat production [20]. It is also indicated that wastewater can contain many contaminants such as drugs, antibiotics, disinfectants, metals and other antimicrobial compounds. These compounds can be found in surface water, in soil and groundwater at different levels of concentration depending on the source and their behavior (degradation rate and adsorption) [21]. Their release into the natural environment is likely to trigger resistance in bacteria [22].

Antimicrobial resistance can be intrinsic or acquired. In the latter case, resistance can arise from mutation of bacterial DNA or from the acquisition of resistance genes by means of horizontal gene transfer, with DNA passing from one bacteria to another [23-25]. It is also indicated that the release of hospital wastewater in the natural environment is a source of both antibiotics and antibiotic resistance genes in groundwater, based on their spatial spreading [26]. In addition, it has been indicated anthropogenically impacted surface waters are an important reservoir for multidrug-resistant bacteria including extended spectrum beta lactamase-producing microorganisms, and antibiotic-resistant genes [27].

The antibiotic inhibition diameters against the wells strains varied from 6.66mm (Amoxicillin+Clavulanic acid against *E. americana*) to 32.66mm (Cefotaxim against *E. coli*). This suggests bacterial resistance in some cases. Groundwater contamination can sometimes result from human impact. In most countries in the world, domestic wastewater, hospital wastewater and slaughterhouse wastewater are sometimes discharged into the environment without any prior treatment. They often contains a variety of antibiotic resistance genes [28]. These wastewaters, with all their bacterial and non-bacterial contents, added to rainwater which contains a diversity of airborne biological particles (also denoted as bioaerosols) and resistance genes, can then contaminate surface water and groundwater [25, 29, 30]. Contamination of surface water could induce interactions firstly between the chemical molecules of spilled wastewater and those of rivers and streams, which could thus lead to the formation of new chemical complexes. Secondly, there could be interactions between these newly formed chemical complexes or the arriving chemical molecules and the bacterial flora. Regarding the contamination of groundwater, water during its infiltration could carry various molecules and bacteria into the groundwater. The degree of this groundwater pollution depends on the self-purifying power of the soil surrounding the water table [31, 32].

Relative differences amongst antibiotics activities have been noted. Those antibiotics mainly act at the cell wall level. The bacterial cell wall, which is located at the periphery of Gram-positive bacteria and within the periplasm of Gram-negative bacteria, comprises a glycopeptide polymer synthesized through cross-linking of glycans to peptide stems on alternating saccharides, which is known commonly as peptidoglycan. Cell wall formation, recycling, and remodelling require numerous enzymes, including a family of enzymes with similar active site character despite distinct and sometimes overlapping roles as carboxypeptidases, endopeptidases, transpeptidases, and transglycosylases, known as "penicillin-binding proteins" (PBPs). The number of PBPs differs between bacteria, in which some are considered essential and others redundant. In general, inhibition of one or more essential PBPs results in impaired cell wall homeostasis, loss of cell integrity, and is ultimately bactericidal [33, 34].

Ceftriaxon acts by inhibiting the mucopeptide synthesis in the bacterial cell wall [35]. The beta-lactam moiety of Ceftriaxon binds to carboxypeptidases, endopeptidases, and transpeptidases in the bacterial cytoplasmic membrane. These enzymes are involved in cell-wall synthesis and cell division. Binding of Ceftriaxon to these enzymes causes the enzyme to lose activity; therefore, the bacteria produce defective cell walls, causing cell death [35].
Cefotaxim inhibits bacterial cell wall synthesis by binding to one or more of the PBPs which in turn inhibits the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls, thus inhibiting cell wall biosynthesis. Bacteria eventually lyse due to ongoing activity of cell wall autolytic enzymes (autolysins and murein hydrolases) while cell wall assembly is arrested [36]. Cefotaxim has activity in the presence of some beta-lactamases, both penicillinases and cephalosporinases, of gram-negative and gram-positive bacteria [36].

Ceftazidim exhibits its bactericidal effect primarily through direct inhibition of specific PBPs in susceptible bacteria [37, 38]. Clavulanic acid binds and inhibits beta-lactamases that inactivate Amoxicillin resulting in Amoxicillin having an expanded spectrum of activity. Amoxicillin inhibits bacterial cell wall synthesis by binding to one or more of the PBPs which in turn inhibits the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls, thus inhibiting cell wall biosynthesis. Bacteria eventually lyse due to ongoing activity of cell wall autolytic enzymes (autolysins and murein hydrolases) while cell wall assembly is arrested [4]. The relative variations in the action mechanisms of antibiotics on the one hand and the intrinsic defense potential of each bacterial strain on the other hand could partly be at the origin of the relative differences observed amongst antibiotics activities at the phenotypic level.

Concerning the physicochemicals of water samples, the pH of the 4 biotopes sampled overall is between 5.28 and 7.4, and the electrical conductivity varied from 180 to 1060 µS/cm. Although the relationship between the abiotic properties of the water sampled and the phenotypic properties of the isolated bacteria has not been assessed, it is known that some water chemical characteristics can significantly impact some bacterial susceptibility against some drugs. For example, significant relationship has been indicated between the pH of the stream water and the *Aeromonas hydrophyla* susceptibility against Oxacillin [39]. According to Chaturvedi *et al* [27], some abiotic parameters of water such as Biochemical Oxygen Demand, Chemical Oxygen Demand, pH and the dissolved oxygen content can be significantly correleted to some resistance genes. An *et al* [40] also noted that the nutrients concentration in an aquatic environment impacts the selection of antibiotic resistance bacteria and exchange of antibiotic resistance genes.

**5. Conclusion**

Wells, stream, hospital wastewater and slaughterhouse wastewater in Butembo (DR Congo, Central Africa) contains enterobacteriaceae including *Klebsiella pneumoniae*, *Escherichia coli*, *Salmonella typhi* and *Ewingella americana*. Their presence or absence frequency varied with respect to the aquatic biotope and bacterial species considered. The antibiotic inhibition diameters and the MICs values of Ceftriaxon, Ceftazidim, Cefotaxim and Amoxicillin/Clavulanic acid varied depending on the antibiotics, the bacterial species and the type of aquatic environment hosting the microorganism. Most of the high MICs and microbial resistance against the four antibiotic were recorded with strains from hospital wastewater. Significant differences in antibiotic inhibition diameter has been noted in most cases amongst bacteria for each antibiotic and each aquatic biotope. The relative variations in the action mechanisms amongst antibiotics on the one hand and the intrinsic defense potential of each bacterial strain on the other hand, as well as the potential influence of the physicochemical properties of each water medium, could partly be at the origin of the relative differences observed at the phenotypic level. It seems necessary to explore the diversity, similarities and differences amongst antibiotic resistance genes in these 4 types of aquatic biotopes.

**Compliance with ethical standards**

*Disclosure of conflict of interest*

No conflict of interest to be disclosed.

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