Carbapenemase-producing Aeromonas species isolated from the urban-impacted Akaki river in Ethiopia

Aeromonas are emerging opportunistic pathogens and are responsible for clinical manifestations such as cellulitis, gastroenteritis, urinary tract infections, as well as life-threatening meningitis and septicaemia (Lamy et al. 2009). Human infections caused by Aeromonas most commonly occur in community settings where they are associated with food-borne disease outbreaks (Batra et al. 2016). Recent reports indicated that Aeromonas is also becoming an important bacterial pathogen associated with wounds related to exposure to contaminated environmental or hospital waters (Rutteman et al. 2017). Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii are the major human pathogens (Elorza et al. 2020). There are a few reports showing the importance of Aeromonas infections among patients with underlying diseases such as cancer. A study from an Ethiopian referral hospital reported that Aeromonas spp. were among the predominant opportunistic pathogens that cause bloodstream infections in cancer patients (Arega et al. 2017). Although its prevalence is low, there was also a report showing Aeromonas-associated camel subclinical mastitis in Ethiopia (Alebie et al. 2021).

The rapid increase of antibiotic resistance in Aeromonas spp. is of serious concern. Aeromonas spp. are becoming increasingly resistant to β-lactam antibiotics, including carbapenems and third- and fourth-generation cephalosporins (Li et al. 2015). Carbapenems are the last-resort antibiotics used to treat infections caused by multidrug-resistant pathogens. However, due to emerging carbapenemase-producing pathogens, their efficacy is increasingly compromised. Aeromonas spp. carry a variety of antimicrobial resistance determinants including β-lactamase-encoding genes. Carbapenem resistance in Aeromonas is commonly mediated by a metallo-β-lactamase (CphA), encoded by the cphA gene that is located on the chromosome and is species-specific (Wu et al. 2012).

Research on antimicrobial resistance has been primarily focused on clinical settings and the role of the environment in the spread and persistence of resistant bacteria is still in its infancy. Faecal contamination is the major source of antibiotic resistance including resistance to carbapenems in the aquatic environment (Mushi et al. 2021). Thus, rivers in megacities serve as potential reservoirs of antibiotic-resistant bacteria, including those resistant to carbapenems. Assessing the distribution of carbapenem resistance in the environment is important to public health since carbapenem is increasingly used as an empirical therapy to treat water-related severe skin and soft tissue infections (Fish 2006). Aeromonas spp. isolated from clinical and environmental samples resistant to certain antibiotics such as β-lactam antibiotics have been reported (Li et al. 2015). There are few reports regarding the resistance prevalence to carbapenems and carbapenemase production (Igbinosa et al. 2017). However, there are no reports in Ethiopia showing the resistance patterns of Aeromonas spp. despite the increasing use of carbapenems in health facilities (Tekele et al. 2021). We previously found that Aeromonas spp. were positively correlated with the diversity and abundance of antibiotic resistance genes (ARGs) in the Akaki river using high-throughput DNA qPCR arrays (data are not shown). Determining the phenotypic resistance and its mechanism in Aeromonas isolates from the aquatic environment gives basic information on how the environment contributes to the spread and persistence of resistant strains. In the current study, we hypothesized that Aeromonas isolates from the aquatic environment are resistant to commonly used antibiotics and the resistance pattern is similar across the different species. We investigated the phenotypic carbapenem and cephalosporin resistance patterns and carbapenemase production in 144 Aeromonas isolates from the Akaki river system, Addis Ababa, Ethiopia.

The study was conducted in Addis Ababa, the capital city of Ethiopia with an estimated population of 5 million. There is a major river running through the city, with two tributaries, the Little Akaki River (locally known as Tinishu Akaki River) and the Greater Akaki River (locally known as Tiliku Akaki River) (Figure 1). Five sampling sites were chosen based on different associated anthropogenic activities such as restricted human activities, irrigation, residential, industry and health facilities. The Ethiopian Public Health Institute had previously selected several of the same sampling sites for water quality surveillance of the Akaki river (EPHI 2017). The sampling sites include Gefersa (GE), Mekanissa (MK), Batu (BA), Zewditu (ZE) and Aba-Samuel (AB). The farthest upstream (GE) and downstream (AB) sites are in areas with fewer anthropogenic activities, whereas the remaining three sites (MK, BA and ZE) are highly impacted by human activities. GE is a drinking water reservoir for the Addis Ababa residents and is farthest upstream of the Little Akaki River. MK is in an irrigation and residential area with small-scale farms that release untreated waste into the river system or its tributaries. BA is in an industry-dominated area along an irrigation zone. ZE is located along healthcare facilities such as referral and primary healthcare hospitals and a densely populated residential area in the Greater Akaki river. AB is the downstream reservoir where both Akaki rivers converge.

Water samples were collected from the five sites during the dry (January) and wet (August) seasons in 2019–2020 for isolation and characterization of Aeromonas species. The dry season in Ethiopia, locally known as Bega, is from October to January and is characterized by hot dry days with an average temperature range of 8.4–21.8 °C in Addis Ababa. The wet season, locally known as Kiremt, is from June to September with an average temperature range of 10.8–19.2 °C and it is when most of the country's food crops are produced. The average rainfall in August was 303 mm (WeatherAtlas 2019/2020). A one-liter (1 L) water sample was collected from each site at 15–20 cm below the surface of the water in sterile bottles in the flow of the river. Samples were placed in a cool box and transported to Armauer Hansen Research Institute (AHRI), Addis Ababa for immediate processing.

Bacteria were isolated using the membrane filtration technique as previously described (WHO 1998). Due to the high level of debris, the water was prefiltered through 25-μm isopore polycarbonate filter and then serially diluted (10-fold serial dilution with sterile distilled water). The diluted water was filtered again through 0.45 μm isopore polycarbonate filter according to the standard protocol and placed on Thiosulfate Citrate Bile-salt Sucrose agar (Sigma–Aldrich) and MacConkey agar (Sigma–Aldrich) and incubated for 24 h at 37 °C. Oxidase-positive single colonies were selected for identification.

Identification was performed by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany). Overnight grown bacterial colonies were applied to disposable MALDI target plates and combined with the matrix (α-Cyano-4-hydroxycinnamic acid (CHCA) (Bruker Daltonik GmbH, Bremen, Germany) in acetonitrile (50%) (Sigma–Aldrich) and 2.5% trifluoroacetic acid (Sigma–Aldrich) according to manufacturer's protocol. The plates were shipped to Orebro University, Sweden for identification.

Identified Aeromonas isolates were tested for their resistance to 10 antibiotics belonging to carbapenems and cephalosporins. The antibiotics tested were the following three carbapenems: ertapenem (ETP_10μg), imipenem (IMP_10μg), and meropenem (MEM_10μg) and the following seven cephalosporins: cefepime (FEP_30μg), cefixime (CFM_30μg), cefotaxime (CTX_30μg), cefotetan (CTT_30μg), cefoxitin (FOX_30μg), ceftazidime (CAZ_30μg) and ceftriaxone (CRO_30μg) (Oxoid, UK). Antibiograms were done on Mueller Hinton agar plates with the Kirby–Bauer Disk Diffusion Susceptibility Test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard procedures to determine the zone of inhibition. Escherichia coli ATCC 25922 was used as a standard quality control strain. Interpretation of results was done using the EUCAST standards (EUCAST 2019). All strains showing ‘resistant’ or ‘intermediate’ characteristics were included under the category ‘resistant’ and the others were classified as ‘sensitive’.

Potential carbapenemase-producing isolates that showed phenotypic resistance to at least one of the tested carbapenems (imipenem, meropenem or ertapenem) were tested for carbapenemase production using the Rapid CarbaNP test (bioMérieux, Marcy Étoile, France) according to the manufacturer's recommendations. It is a hydrolysis-based assay for rapid qualitative detection of carbapenemase enzyme in bacteria with 99.6 and 97.4% sensitivity and specificity, respectively (Garg et al. 2015). Freshly grown colonies on Muller Hinton agar (Oxoid, UK) were used for the test.

Statistical analyses were performed using SPSS software version 20.0 (IBM, USA). Descriptive statistics were presented as numbers and percentages. Statistical association of the difference in the susceptibility patterns and season of isolation was determined by χ² test or Fisher's exact test as appropriate. Binary logistic regression was used to determine the differences in the resistance pattern of isolates and sampling sites.

A total of 163 Aeromonas isolates were recovered from the five sampling sites of the Akaki river. The major clinically relevant Aeromonas spp. detected were A. caviae (62), A. hydrophila (33) and A. veronii (49) (Table 1). A. caviae was predominantly isolated in the anthropogenically impacted sites (BA, MK and ZE), whereas 37% of A. veronii were detected in the farthest upstream site (GE) (Table 2). These three Aeromonas spp. commonly implicated in human infections were tested for their resistance patterns to carbapenem (ertapenem, imipenem and meropenem) and cephalosporin (cefepime, cefixime, cefotaxime, cefotetan, cefoxitin, ceftazidime and ceftriaxone) antibiotics. The highest rate of resistance was observed against ertapenem 71 (49.3%), imipenem 65 (45.1%), cefixime 76 (52.8%), cefotaxime 86 (59.7%) and ceftriaxone 79 (54.9%) (Table 3).

Carbapenem antibiotics are an important treatment option for life-threatening infections. However, the evolution and spread of carbapenem-resistant bacterial pathogens are steadily increasing and becoming a major global public health threat (Adams et al. 2017). In the current study, the isolates demonstrated a high rate of resistance to the following three tested carbapenem antibiotics: ertapenem 71 (49.3%), imipenem 65 (45.1%) and meropenem 53 (36.8%) (Table 3). Comparable results have recently been reported by Wang et al. (2021) where 54.96 and 46.56% of the clinical Aeromonas isolates were resistant to imipenem and meropenem, respectively. However, contrary to our results, very few were resistant to ertapenem (Wang et al. 2021). This may be due to increased prevalence of resistant strains in the community, thus factors such as increased faecal pollution, antibiotic residues, and other pollutants such as heavy metals may exacerbate the problem. In addition, horizontal gene transfer between the bacterial communities may also play a significant role in the spread of resistant genes between the bacteria in aquatic environments where selection pressure is present (Baker-Austin et al. 2006).

A distinct level of resistance to carbapenem antibiotics was observed among the different Aeromonas spp. The majority of A. hydrophila and A. veronii were resistant to all three carbapenem antibiotics tested (ertapenem, imipenem and meropenem), with the highest being among A. veronii isolates (Table 3). The prevalence of resistance of A. veronii to ertapenem, imipenem and meropenem was 87.8, 75.5 and 63.3%, respectively. None of A. caviae isolates were resistant to imipenem and ertapenem and only 4.8% were resistant to meropenem. Cross-resistance in carbapenem antibiotics was also found with 54 isolates resistant to both imipenem and ertapenem, 44 isolates to meropenem and imipenem and 47 isolates to ertapenem and meropenem. It has been reported that carbapenem resistance in Aeromonas is commonly mediated by metallo-β-lactamase (CphA), encoded by the bla_cphA gene, and the distribution is species-dependent. The gene is frequently found in A. hydrophila and A. veronii, but not in A. caviae (Wu et al. 2012) and therefore, the observed low prevalence of resistance in A. caviae to carbapenem antibiotics in the current study could be due to the absence of bla_cphA gene.

The prevalence of Aeromonas spp. resistant to carbapenems in the current study was higher than previously reported in clinical isolates (Rosso et al. 2019). Furthermore, none of the Aeromonas strains isolated from cholera-like illnesses were resistant to meropenem (Mohan et al. 2017). This indicated that the environmental factors such as the presence of diverse bacterial communities and chemical pollutants may provide selection pressure favouring the persistence of these carbapenem-resistant strains. In a similar study, antibiotic resistance was significantly higher in Aeromonas isolated from environmental water than those from clinical samples. For instance, resistance to imipenem was 22 and 18% for environmental and clinical isolates, respectively (Li et al. 2015). The high prevalence of resistance to ertapenem in the current Aeromonas isolates was comparable to a report by Igbinosa et al. (2017), where all the environmental isolates were resistant to ertapenem (Igbinosa et al. 2017).

The major resistance mechanism to ertapenem in Enterobacteriaceae is due to the expression of β-lactamases such as AmpC β-lactamase or extended-spectrum β-lactamase (ESBL) combined with porin loss (Hawser et al. 2012). Therefore, the Aeromonas isolates that were phenotypically resistant to carbapenem antibiotics were tested for carbapenemase production. The majority of A. hydrophila (82.1%) and almost all A. veronii isolates (94.4%) that were resistant to at least one of the tested carbapenem antibiotics produced carbapenemase, however, the few A. caviae tested were carbapenemase non-producers (Figure 2). The presence of carbapenemase-producing Aeromonas in the Akaki river is a public health concern as the water is used to grow vegetables that are eaten raw and for domestic purposes in the downstream community. Farmers using the water for open irrigation are easily exposed to the polluted water and often complain of skin diseases and wound infections (Woldetsadik et al. 2018).

Considerable numbers of the Aeromonas isolates were also resistant to cephalosporins, with a high prevalence to the third-generation cephalosporins, including cefotaxime at 59.7%, followed by ceftriaxone (54.9%) and cefixime (52.8%). The prevalence of resistance to cephalosporin in the current study is higher than the report in South Africa in Aeromonas isolates from surface water and waste water (Govender et al. 2021). This discrepancy could be due to the different sources of pollution, or the inclusion of isolates collected during different seasons in the current study. The majority of A. hydrophila and A. veronii isolates were susceptible to cephalosporins compared to A. caviae. For instance, resistance to cefixime was 83.9% (52/62), 36.4% (12/33) and 24.5% (12/49) for A. caviae, A. hydrophila and A. veronii, respectively. The majority of A. caviae and A. hydrophila isolates were cefotetan-resistant, whereas only 7.6% of A. veronii were resistant to cefotetan. The resistance to cephalosporins was higher in A. caviae than in the other two species. For instance, a significant number of A. caviae were resistant to cefixime, cefotaxime, cefoxitin and ceftriaxone. Resistance to cephalosporins in Aeromonas is mediated by class B, C or D β-lactamases (Chen et al. 2012). The difference in the prevalence of resistance to this group of antibiotics among the different species could be due to the presence of species-specific resistance genes as previously reported (Chen et al. 2012). The observed resistance to cephalosporins could also be attributed to the natural resistance of the isolates.

Significant (p<0.05) seasonal variation was observed for resistance to ertapenem, imipenem and meropenem (Table 4, Figure 3). Resistance to these antibiotics was higher during the wet season than the dry season. The seasonal variation could be explained by different factors. For instance, during the wet season, faecal pollution is higher and results in greater contamination of the river by human- or animal-derived bacteria potentially resistant to carbapenems, suggesting community carriage of resistant Aeromonas. Previously, it has been reported that rainfall plays a significant role in the introduction of faecal pollution to the recipient river system (Mushi et al. 2021). Recurring faecal pollution from nearby households, poultry farms and hospitals is also a potential source of already resistant bacteria, and this increases the concentration of resistant strains by providing human-derived Aeromonas strains to the pool. However, previously, the highest prevalence of carbapenem-resistance was reported during dry, and not the wet season (Diwan et al. 2018). This could be due to the different bacterial species included in the studies, since Diwan et al. (2018) reported E. coli whereas the present study focused only on Aeromonas spp. Previously it has been reported that the differences in bacterial strains leads to variation in the prevalence of resistance in different seasons (Gönder et al. 2021).

The relationship between season and antibiotic resistance in the present study was not consistent across the tested classes of antibiotics. For cephalosporin antibiotics such as cefepime, cefixime, cefotaxime, cefotetan, cefoxitin, ceftazidime and ceftriaxone, resistance was higher during the dry season than the wet season (Table 4, Figure 3). One of the main factors influencing persistence of antibiotic-resistant bacteria in the aquatic environment is the continual discharge of antibiotics and pharmaceuticals into the water system and this would result in different concentrations in different seasons. The main differences between the seasons are water level, temperature and flow, and it has been reported that antibiotic concentrations tend to increase during the dry season. However, it also depends on the chemical nature and degradability of the antibiotics in the natural environment. Another factor that may contribute to the varying antibiotic resistance is the difference in the consumption of antibiotics during the two seasons and this may affect their level in the environment. It is therefore plausible that the higher prevalence of resistance to cephalosporins in the dry season may be associated with increased selection pressure caused by an increased concentration in the water. In a previous study, it has been observed that E. coli isolated from activated sludge and secondary treatment effluent had significantly higher resistance to amoxicillin in summer than winter (Honda et al. 2020). Another report from Cali City, Colombia showed that resistance to cephalosporins, especially against third-generation cephalosporins, among Enterobacteriaceae isolates from water sources was more prevalent during the dry season (Chavez et al. 2019). Our results are consistent with a previous report where significantly higher levels of resistant isolates were found during the dry season (Chavez et al. 2019). However, in a study from India, there was no significant difference in resistance to the majority of cephalosporins studied in E. coli isolated during different seasons (Diwan et al. 2018). The variations in the patterns of resistance against the two classes of antibiotics during the wet and dry seasons may also be associated with the different chemical stability in the water or changes in the composition of the Aeromonas species. It was recently reported that Enterobacteriaceae isolates had varied resistance patterns to different antibiotics, where resistance to cephalosporin antibiotics such as ceftazidime was higher during the dry season and resistance to ciprofloxacin was significantly higher during the wet season (Díaz-Gavidia et al. 2021). The data in the current study presented results from a 1-year sampling, therefore a longitudinal study over more years would be necessary to discriminate the mechanisms behind the antibiotic resistance variation during the seasons. The increased prevalence of resistant isolates during the dry season in the Akaki river system is a significant concern as the water is extensively utilized for domestic purpose during this season due to water shortage.

Significant spatial variations of resistance patterns in Aeromonas among the different sampling sites were observed. Most of the isolates in the farthest upstream (GE) and downstream (AB) sites were susceptible to cephalosporin antibiotics compared to anthropogenically impacted sites (Figure 4). For instance, isolates from MK and ZE sites were significantly more likely resistant to cefotaxime compared to isolates from the farthest upstream (GE) with a 95% CI of 6.14 (1.60–23.50) and 22.75 (5.56–93.17), respectively (Supplementary Table S1). The increased resistance patterns in these two sites may be due to contamination by human-derived strains as the two sites are in residential areas, and near healthcare facilities (ZE). The presence of irrigation at the MK site is also a possible environmental factor for emergence of resistant strains, as previous reports show pesticides and fertilizers could trigger the persistence of resistant strains due to cross-resistance between pesticides and antibiotics (Ramakrishnan et al. 2019). Similarly, the isolates from the three anthropogenically impacted sites were highly resistant to ceftriaxone. Previous reports have shown a positive correlation between anthropogenic activities and patterns of antibiotic resistance and ARGs (Zhang et al. 2022). Unlike the cephalosporin antibiotics, isolates from the GE reservoir were more likely resistant to carbapenem antibiotics than those from the other sites. The variation of resistance patterns among the sampling sites may be due to the differences in exogeneous input or difference in the Aeromonas species, which have different resistance patterns. For instance, V. veronii had the highest level of resistance to carbapenem antibiotics and it was frequently detected in GE when compared to the other sites. Since the GE reservoir is restricted to human activities, the high prevalence observed could be associated with the existence of naturally resistant Aeromonas spp. or contamination by wild animals, including fish as Aeromonas are primary fish pathogens (Soto-Rodriguez et al. 2018). The presence of resistant Aeromonas to last-resort antibiotics in the drinking water reservoir is of great public health concern.

In conclusion, we report that the Akaki river water in Addis Ababa harbours a high abundance of resistant Aeromonas isolates, some of which are potential human pathogens. It was observed that carbapenem resistance among some of the Aeromonas spp. was mediated by carbapenemase production and the prevalence of resistance to carbapenem antibiotics is species-dependent, with the lowest resistance rate in A. caviae. Furthermore, there was a higher frequency of carbapenem resistance in wet season, whereas a higher frequency of cephalosporin resistance was found in the dry season. However, the association of antibiotic resistance between the wet and dry seasons in the present study was not consistent across all the antibiotics tested which could be associated with both environmental and anthropogenic activities.

The study was approved by the Addis Ababa University institutional research board, College of Health Sciences (IRB-CHS; Ref. No. AAUMF 03-008), Armauer Hansen Research Institute/All Africa Leprosy TB Rehabilitation and Training center (AAERC) research ethics committee (Ref. No. PO26/17) and national IRB (Ref. No. 310/83/2018). Permission for sample collection was obtained from the Addis Ababa Water and Sewerage Authority. Ethiopian Biodiversity Institute authorized the exportation of isolates to Örebro University, Sweden.

B.Y., J.J., P.E.O., Y.W., D.A., A.M. and A.A. designed the study. B.Y. performed the experiments. B.Y., Y.W., D.A., J.J., and P.E.O. analyzed the data and wrote the initial draft of the manuscript. All authors contributed to the revisions of the manuscript and approved it for publication.

This study was supported by the Swedish International Development Agency (SIDA) through its support to the Armauer Hansen Research Institute (AHRI), Svenska Forskingsrådet Formas (Grant number: 219-2014-837; J.J.), Örebro University and Addis Ababa University.

All relevant data are included in the paper or its Supplementary Information.

The authors declare that they have no conflict of interest.