Acinetobacter baumannii has emerged over the few last decades as a cause of healthcare-associated infections with this organism associated with increased patient morbidity, mortality, and treatment costs.1–3 Its clinical significance has been propelled by its remarkable ability to upregulate or acquire resistance determinant making it one of the organisms threatening the current antibiotic era.4,5 The possession of this array of resistance mechanisms have made this organism to be able to resist almost all available antibiotics in hospital environment and has position the organism as important candidate for the evaluation of reservoirs of antibiotic resistance in the environment or even in human subjects.6Acinetobacter. baumannii have the ability to survive in both moist and dry conditions for many weeks or even months, hence this organism is widely distributed in natural and nosocomial environments, human skin, as well as mucosal surfaces facilitating its spread.7–9 The cross-transmission of this organism from patient to patient and the possibility of outbreak extension by patient transfer have been demonstrated.10 Despite hospital ecology being intensively studied, its ecology outside the hospital remains unclear or poorly defined impeding effective prevention of transmission.2,11 Several investigators have suspected that the survival of A. baumannii in the environment (in particular in water) could contribute to the transmission of the organism during outbreaks.12 It is well established that members of the genus Acinetobacter is ubiquitous but difficulty in unequivocally identify this organism has made the ubiquity in nature of A. baumannii a widespread misconception.1,9 Therefore the existence of an extra-hospital reservoir of A. baumannii and the implication of this potential reservoir in the occurrence of certain infections due to this organism are still controversial.1,2 Additionally, the prevalence and antimicrobial resistance of A. baumannii outside the hospital setting has been poorly investigated.13 There seems to be a continuous influx of novel strains into the clinical setting with the potential of new infectious features.11 Studies have shown that infections caused by extra-hospital strains of A. baumannii are responsible for community-acquired infections mainly in tropical and subtropical areas.5,14 This suggests a source of this pathogen outside of the hospital and therefore the significance of environmental isolates in the epidemiology of A. baumannii is of a great concern worldwide. There is no clear evidence about the way of introduction of A. baumannii into hospital environment, its propagation from hospital settings to the natural environment and its natural habitat outside hospitals.15 Also unlike clinical strains, there are limited reports using molecular analysis to explore the extra-hospital epidemiology of A. baumannii.11,16 The presence of A. baumannii in the aquatic environment raises the issue of public health risk since people are in constant contact with the aquatic environment.17 Hospital waste water is discharged into municipal sewage system without prior treatment, contributing to widespread contamination of natural waters with emerging pathogenic bacteria that can carry MDR genes.18 In the aquatic environment, bacteria interact with various organisms, some of which are fish used for consumption as well as commercial or recreational purposes.19 Most of the antibiotics used in the hospitals end up contaminating our local dams. The indiscriminate use of antibiotics in livestock production as growth promoters or to control infectious diseases and the treatments have been linked with the dissemination of resistant bacteria which can be transferred to people via food, direct contact with infected animal or through the abattoir. In a study conducted in Scotland, pigs and cattle slaughtered for human consumption and coming from different farms were sampled (faeces, skin, nostril and ear) for A. baumannii isolation. The prevalence of A. baumannii carriage was 1.2%. The 16 A. baumannii isolated were grouped into three different clusters, but had different pulsed-field gel electrophoresis (PFGE) patterns compared with human isolates of the three major European clones Eci, ECII, and ECIII.20 Recent reports have also described the presence of carbapenem-resistant A. baumannii in animals. The OXA-23 carbapenemase has been found in Acinetobacter from cattle, horses, and cats.21–23 The NDM-1 has also been reported in Acinetobacter spp. from food animals (chicken and pig farms) in China.24,25 However, knowledge about carbapenemase-producing A. baumannii of environmental origin (aquatic and abattoir in case of this study) remains very limited, making it difficult to assess its impact on public health. In South Africa, A. baumannii has been isolated from hospital environments; nevertheless only few studies have reported the presence of A. baumannii in the aquatic environment, meat of food animals and the environment where these meats come from. These areas are a source of water and food and therefore can be of transmission of this organism into hospital settings or may horizontally transfer resistance genes to other organisms.

Ethical approval for this project was obtained from the Walter Sisulu University Research Ethics and Biosafety Committee [Reference number: 019/2016]. Also written permission was sought from the authorities of the two study sites. For the protection of animals used for scientific purposes the experiments described complied with the guidelines of the European Union Council http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm (Directive 2010/63/EU).

The data were analyzed using the Statistical Package for Social Sciences (SPSS, IBM, version 23.0 Armonk, NY). The frequency of some parameters including A. baumannii susceptibility and also resistant genes were compared between abattoir and aquatic A. baumannii by Chi-square or Fisher’s exact test. P-values <0.05 were considered as significant.

Over the past two decades, Acinetobacter species have become virulent pathogens, responsible for nosocomial infections and outbreaks, particularly in intensive care units and high dependency units (HDUs).2 Majority of published studies have concentrated on the hospital epidemiology of these organisms2 and animal health care settings making it difficult to demonstrate the extra-hospital origin of A. baumannii. The intensive use of antibiotics over the last few decades in human and as growth promoting and as prophylactic agents in livestock have resulted in serious environmental and public health problems, since this enhances antimicrobial selective pressure. Also anthropological activities have great impacts on our local dams. The antibiotic residues concentration increases in hospital and industrial effluents and these end up in our local dams leading to the development of antibiotic-resistant bacteria. Gros et al., studied the antibiotic concentrations in aquatic environments and the median concentrations in surface and ground water were reported as 0.030 and 0.071 µgL−1, respectively.40 These concentrations are above the MIC/MBC for all antibiotics, according to the British Society for Antimicrobial Chemotherapy.41A. baumannii isolated from various environmental locations has been linked with nosocomial spread.42 The resistant bacteria from the extra-hospital environments may be transmitted to humans, in whom they cause disease that cannot be treated by conventional antibiotics. In aquatic environment two hypotheses can arise from the detection of A. baumannii: (1) water is a normal habitat of A. baumannii, or (2) the presence of the bacterium results from human or animal contamination. To our knowledge, this is the first study investigating the molecular mechanisms accounting for the multidrug-resistance observed in A. baumannii recovered from abattoir and aquatic samples in the Eastern Cape Province of South Africa.

In the current study, the prevalence of A. baumannii was 14.2% in abattoir and 14.0% in aquatic samples collected. Of these, MDR A. baumannii was recovered from 56.5% (48/85) abattoir isolates and 53.1% (52/98) aquatic isolates and were analyzed. Therefore, our study evidenced the extra-hospital presence of A. baumannii in Eastern Cape Province of South Africa. This is in agreement with a previous study that found 51.2% (85/166) from meat samples to be MDR,9 i.e. acquired resistance to at least one agent in three or more antimicrobial categories among the eight different classes of antibiotics used to define MDR.29 In contrast, a study conducted by Stenstrom et al. in Nkonkobe Municipality of the Eastern Cape Province, South Africa, found that the prevalence of Acinetobacter calcoaceticus complex from soil and water samples in Alice town was 41%.6 Also previous studies in Senegal, Scotland, and La Reunion Island isolated A. baumannii as animal colonizers in 5.1%, 1.2%, and 6.5%, respectively.5,13,20 The study also revealed the quantitative changes in antibiotic resistance at two sampling sites. In general, susceptibility testing showed that 98% in abattoir isolates and 90% in aquatic isolates were resistant to ampicillin which is usually a non-effective antibiotic for the treatment of Acinetobacter infections. A high incidence of ampicillin resistance in aquatic environments was reported.43 High resistance to trimethoprim/sulfamethoxazole in abattoir (100%) and aquatic isolates (92%) is a result of the inherent resistant of A. baumannii to this compound through the target protein, dihydrofolate reductase, which has a low affinity for the drug44 and also to selective pressure caused by the use of these antimicrobial agent for treatment of infections in cattle. The MARI is a good risk assessment tool and its value (nominally 0.20) has been applied to differentiate low- and high-risk regions where antibiotics are overused.45 The present study showed MARI ranging from 0.20 to 0.52 (mean MARI; aquatic, 0.28 and abattoir, 0.34) which indicates that the isolates emerged from high-risk sources of contamination and also gives an idea of the number of A. baumannii isolates showing antibiotic resistance in the risk zone of the susceptibility study. This indicates that the study areas where these isolates came from were exposed to an environment of high-risk contamination from a region or area where there is high antibiotic use,30 probably from hospital and industrial effluents which find their way into aquatic environment or the indiscriminate use of antibiotics in the livestock slaughtered at the abattoir. This can lead to high antibiotic resistance selective pressure.46 The high resistance rate to ciprofloxacin, gentamycin, and tetracycline was not expected. These antimicrobials may have been used in these animals either as growth promoter, prophylactic or for treatment in case of abattoir isolates and also ending in aquatic environment. This result reflects the use of antimicrobial agents in livestock in South Africa, as tetracycline has long been the antimicrobial most commonly used in livestock, accounting for over 30% of the total amount of antimicrobial consumption.47 Furthermore the high resistant rate to cephalosporins may be because this antibiotic is frequently used in hospitals and might have found their way into the aquatic environment in case of the isolates from the dam or might have been introduced as a growth promoter, prophylactically or for treatment of food animals that were slaughtered at the abattoir. The current study showed that all isolates from both sources were susceptible to colistin and tigecycline which are last line antibiotics. Colistin and tigecycline are normally used as a drug of last resort in hospitals which limits the possibility of bacteria developing resistance against these antibiotics. Also, they are rarely used in animals either as a growth promoter, prophylactically or for treatment. It is also noteworthy to highlight the fact that there was no statistical difference in the resistance rates of A. baumannii isolates recovered from the abattoir and from the aquatic environment (p < 0.253). This wide range of resistance, as demonstrated here, is a cause for concern.

In this study, isolates were also tested molecularly for gene similarities. Molecular analysis of the 48 and 52 MDR A. baumannii isolates showed that all isolates from both sources harboured blaOXA-51–like and 32 (66.7%) and 11 (21.2%) isolates harboured blaOXA-23–like genes in abattoir and aquatic isolates, respectively. This indicates that blaOXA-23 was the most prevalent carbapenem hydrolyzing class D β-lactamase gene frequently found in carbapenem-resistant A. baumannii isolates collected from extra-hospital environments. Our study showed that blaOXA-23 producers in particular and carbapenemase producers in general, may be isolated from extra-hospital environments. Among the hypotheses that could explain the selection of this carbapenemase is the indiscriminate use of penicillins or penicillin–β-lactamase inhibitor combinations. This could create selective pressure for β-lactamases because blaOXA-23 does confer, in addition to decreased susceptibility to carbapenems, a high level of resistance to those compounds. It is well established that animals can be reservoirs of antimicrobial-resistant bacteria.48 These identified determinants are not only of concern in A. baumannii. Rather, these genes can be transferred to other bacteria by horizontal gene transfer and are of great concern to human health. Therefore, there is the need to emphasize on strict adherence to personal and general hygiene to reduce the risk of opportunistic infection by A. baumannii which is difficult to control. Cases of Acinetobacter infection have been traced to environment due to poor hygiene, especially hand hygiene.1

The current study showed that all MDR isolates lacked significant antibiotic resistance features, such class 1 integrons and ISAba1, indicating that the class 1 integron and ISAba1 associated resistance traits are not implicated in MDR of environmental A. baumannii. This is in agreement with a study conducted by Hamouda et al. where no class 1 integrons and ISAba1 were found in A. baumannii isolates from recently slaughtered animals destined for human consumption and collected at major Scottish abattoirs.20 ISAab1 is considered the first step in resistance evolution in A. baumannii.31 The absence of these resistance-driven mobile elements in these sources suggests that no or little antibiotic selective pressure was applied to these pathogens, contrary to their clinical counterpart. Therefore the high resistance rate to carbapenems which is the drug of choice to treat A. baumannii infections in both abattoir and aquatic isolates can be partly linked to the increased expression level of blaOXA-51-like carbapenemases. In addition, it is speculated that ISAba1 attains full expression under conditions of excessive selective pressure (presence of carbapenems); thus, one would not expect to find this mechanism of resistance in an environmental isolate.14 However, three isolates (AB4, AB28 and AF3) that expressed blaOXA-51-like in our study unexpectedly showed imipenem MICs that were lower than 4 μg/mL, which suggests that imipenem resistance in some A. baumannii strains may be primarily modulated by genes other than blaOXA-51-like. This can also be due to the blaOXA-51-like genes being usually silent or poorly expressed,49 thus not conferring a resistance phenotype. Also, the increased use of carbapenems antibiotics to treat food producing animals may explain the high level of resistance to antibiotics of this class observed in the current study. The present results provide evidence that extra-hospital A. baumannii isolates serve as a reservoir of multiple antibiotics resistant and hence as potential route for the entry of MDR pathogens into human population. Our results also suggest that the nosocomial pathogen A. baumannii is well adapted to different environments, not only to the hospital setting. This has very important implications for human health, as infections by MDR are difficult to treat and often requires expensive antibiotics, long term therapy and even mortality. Further studies should be performed ascertain the prevalence of MDR A. baumannii in other sources.

This study has demonstrated by molecular identification that MDR A. baumannii is actually present outside the hospital. The presence of MDR A. baumannii in the extra-hospital environment may be a threat to public health considering that this may provide a vector for the spread of these opportunistic pathogen into both community and hospital settings environment. Coordinated approaches to reduce integrated human health risks in the environment as well as careful compliance with the WHO guidelines50 on surveillance, rational antibiotic prescribing, and standard treatment guidelines for both community- and hospital-acquired infections will lead appreciably towards reducing the ever-rising threat of antibiotic resistance. However, further studies are needed for better understanding of the mechanism of interactions between the different potential reservoirs and humans. Also, possible impact on the horizontal transfer of blaOXA genes, surviving in selected condition or occurrence of infection outside the hospital setting should be further investigated.

Funding of the research through the National Research Foundation (NRF) Thuthuka grant UID: 107619 is graciously appreciated. Beyond this, it did not take part in the research activities of knowledge creation and dissemination. The Nelson Mandela Academic Hospital (NMAH) and the Microbiology Laboratory at the National Health Laboratory Services (NHLS) at the NMAH are also acknowledged.

All data generated or analyzed during this study are included in this published article (and its supplementary information file).

Ethical approval for this project was obtained from the Walter Sisulu University Research Ethics Committee (Human) [Reference number: 019/2016].

The authors declare that there is no conflict of interests regarding the publication of this paper.