Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa

Background : Several Acinetobacter species live in different ecosystems such as soil, freshwater, wastewater, and solid wastes. In this study, we assessed the occurrence of A. baumannii and A. nosocomialis , which are the major two clinically important species of the genus Acinetobacter , in three freshwater resources (Great Fish, Keiskemma, and Tyhume rivers) in the Eastern Cape Province, South Africa over a one year sampling regime (April 2017 - March 2018). Presumptive Acinetobacter species were subjected to molecular identi�cation by using Acinetobacter genus-specic primers targeting the recA gene. The con�rmed Acinetobacter species were further delineated into A. baumannii and A. nosocomialis using species-specic primer sets. Similarly, virulence genes, namely; afa/draBC, epsA, �mH, OmpA, PAI, sfa/focDE , and traT in the two Acinetobacter species were also determined using molecular techniques. Result : A total of 1107 presumptive Acinetobacter isolates were recovered from the freshwater resources of which 844 was con�rmed positive for the Acinetobacter genus. Of the 844 Acinetobacter isolates, 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great Fish, Keiskemma and Tyhume rivers respectively. Our �nding revealed that 410 (48.58%) and 23 (2.7%) of the isolates were con�rmed to be A. baumannii and A. nosocomalis , respectively. The presence of these clinically-important Acinetobacter species in the freshwater studied suggests possible contamination of the selected rivers and also that A. baumannii and A. nosocomialis can thrive in aquatic environments. Besides, 308 (75.12%) A. baumannii and 3 (13.04%) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p<0.05) virulence gene in A. baumannii with 69 (45.10%), 52 (50.98%) and 77 (49.68%) isolates in Great Fish, Keiskamma and Tyhume rivers respectively. Conclusion : The occurrence of these pathogens in rivers which are consumed by humans and livestock, as well as being used for irrigation system constitutes a risk to public health.


Background
The majorities of Acinetobacter species are free-living and are ubiquitous in nature, but the clinicallyimportant species, most especially the Acinetobacter calcoaceticus-baumannii (ACB) complex, are frequently isolated from the hospital environs.Among members of this complex, A. baumannii, A. nosocomialis, and A. pittii are well-reported causative agents of Acinetobacter-related infections in hospitals around the globe [1,2].The most virulent Acinetobacter species, A. baumannii, is known to cause disease outbreaks in intensive care units (ICUs).Owing to the importance of this disease-causing species in the clinical settings, some scientists have reported that it is a nosocomial pathogen and its occurrence in other environments is likely not possible [2].However, in recent discoveries, A. baumannii has been reported to thrive in freshwater, soil, healthy human skin just like other non-pathogenic species in the Genus [3,4].The occurrence of A. baumannii and other pathogenic species of the genus Acinetobacter in other environment apart from the hospital could be associated with the indiscriminate disposal of wastewater and materials from the hospital into the environment [5].
The pathogenicity of Acinetobacter species like other microorganisms is strongly associated with the virulence factors they harbour.These factors include porins (OmpA), capsular polysaccharides, lipopolysaccharides (LPS), phospholipase, outer membrane vesicles (OMVs), protein secretion systems, metal acquisition system etc.[8, 17,18,19].The OmpA is a protein located on the outer cell membrane of the bacteria, which is responsible for the selective permeation of materials in and out of the cell.The OmpA also binds to the host epithelial cells in order to gain entry into the cell cytoplasmic environment.As such, it causes cell death (apoptosis) by releasing the cytochrome c.OmpA was also noted as one of the factors with which A. nosocomialis initiates its pathogenesis [20].Pathogenic Gram-negative bacteria are known to secret outer membrane vesicles (OMVs) [17,21,22], for interaction between the bacterial pathogens and the host cells [23].Kim and others [20] showed that A. nosocomialis uses its outer membrane vesicles (OMVs) for secretion of cytotoxic factors with which it elicits an immune response from host epithelial cell.Phospholipase, like other factors, also contributes to the virulence of the pathogenic Acinetobacter species, by hydrolysing phospholipid bilayer of the host cell membrane in order to destabilize the entire cell [17,24].
As a result, an investigation of the occurrence and distribution of these clinically-important Acinetobacter species in other environments, other than hospital, is very necessary for the understanding of what nature and diversity truly represent [25,26].As such, the time has come to rede ne the environmental coverage by these organisms, beyond a clinical setting.Therefore, the focus of this study was to assess the occurrence, distribution and the virulence factors of clinically-important Acinetobacter species such as A. baumannii and A. nosocomialis in the selected freshwater resources in the Eastern Cape, South Africa.

Results
Isolation and distribution of presumptive Acinetobacter species A total of 1107 presumptive Acinetobacter species was recovered from the three rivers studied, of which 370, 309 and 428 presumptive isolates belonged to Great Fish, Keiskemma and Tyhume rivers respectively as summarized in Table 1.

PCR ampli cation of the recA gene
To further validate the occurrence and distribution of the bacteria in the genus Acinetobacter in this study, the identi cation was achieved by using the PCR-based assay to detect internal recA genes that are speci c to all Acinetobacter species.Figure S1 presents the PCR product of the gel electrophoresis and staining for the ampli cation of the 425 bp fragment, which correlates to recA gene.Out of the 1107 presumptive isolates, 844 were con rmed to belong to the genus Acinetobacter of which 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great sh, Keiskemma and Tyhume rivers respectively.The con rmed isolates belonging to the genus Acinetobacter is summarized and presented in Table 1.

Delineation of Genus Acinetobacter into species
Figures S2A and S2B shows the PCR products of the gel electrophoresis and staining for the delineation of the genus Acinetobacter into A. baumannii and A. nosocomailis accordingly, while A. pittii was not detected.

Detection of Virulence Genes
The gel electrophoresis of the virulence genes; afa/draBC, epsA, mH, OmpA, PAI, sfa/focDE, and traT, are presented in Figure S3.The virulence genes haboured by A. baumannii and A. nosocomialis in this study is presented in Figure 2 and summarized in Table 2. Additionally, the virulence gene(s) associated with each of the Acinetobacter species as well as the river source from which they were isolated is described.The statistical signi cance of the prevalence virulence genes with respect to rivers was also emphasized.
Three hundred and eight (75%) of the 410 A. baumannii and 3 (13%) of the 23 A. nosocomialis isolated from the rivers were observed to exhibit one or more virulence genes, out of the seven tested.Respectively, 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates from the three rivers did not harbour any of the virulence genes tested in this study.However, there was a signi cant difference (p<0.05) in the prevalence of virulence genes harboured by A. baumannii compared to A. nosocomialis, though the population of the former was signi cantly higher than the latter in all cases.
The prevalence of virulence gene afa/draBC was signi cantly higher (p<0.05) in the isolates from Great Fish river in comparison to Keiskamma and Tyhume rivers.However, there was a signi cant difference (p<0.05) in prevalence between the afa/draBC genes haboured by the bacteria recovered from the Tyhume river when compared to those from Keiskamma river.Besides, the prevalence of traT gene in Acinetobacter species from Great Fish and Tyhume rivers showed no statistical difference (p<0.05),but both were signi cantly higher (p<0.05)than those detected in isolates from Keiskamma river.Similarly, the prevalence of mH gene detected in isolates recovered from Great Fish river was signi cantly higher (p<0.05)than those detected in isolates from both Keiskamma and Tyhune river.Nonetheless, the proportion of isolates harbouring the gene in Keiskamma was signi cantly (p<0.05)lower than those from Tyhume river.The prevalence of the PAI virulence gene was not signi cantly (p<0.05)different in all the rivers studied.In Great Fish and Keiskamma rivers, there was no signi cant difference (p<0.05) in the prevalence of sfa/focDE in isolates recovered from the rivers, but the proportion of Acinetobacter species in Tyhume harbouring the gene was signi cantly higher (p<0.05)than in those from both Great Fish and Keiskamma rivers.The number of Acinetobacter species harbouring epsA virulence gene in the Great Fish river was signi cantly higher (p<0.05)than those recovered from Keiskamma river, but signi cantly lower (p<0.05)than in Acinetobacter species recovered from Tyhume river.The prevalence of the OmpA virulence gene was signi cantly higher (p<0.05) in A. baumannii isolates than all other virulence genes in this study.Besides, OmpA was the most prevalent virulence gene in the rivers, which represented 69 (45.10%), 52 (50.98%) and 77 (49.68%) A. baumannii isolates in Great Fish, Keiskamma and Tyhume rivers respectively.The number of Acienotobacter species harbouring OmpA gene in Great sh river were signi cantly higher (p<0.05)than isolates from Keiskamma river, whereas Tyhume river maintained the highest level of statistical signi cance (p<0.05) of the total Acinetobacter species exhibiting the virulence gene.
Summarily, OmpA was the most prevalent virulence gene detected in Acinetobacter species in all the three rivers studied followed by mH and epsA genes, whereas PAI and Sfa/focDE genes were the least exhibited respectively.Furthermore, the number of Acinetobacter species habouring the highest percentage of virulence genes was isolated from the Tyhume River followed by the Great Fish River, while Kieskamma River was the least.

Discussion
Bacteria in the genus Acinetobacter have been known to colonize a wide array of ecological systems of which water, soil, sludge, wastewater, plants' root, and animals have been prominent.In this study, water samples were collected at ve different sites from three selected rivers in the Eastern Cape Province, South Africa for the assessment of the oof Acinetobacter species.Several presumptive species belonging to the genus Acinetobacter were recovered from the water samples.Similar study was carried out by [27] on the microbial community in the urban riverine environment in Croatia, where 57 of the isolates belong to the genus Acinetobacter.Likewise, Krizova et al. [28], noted a widespread of A. bohemicus in the water environment in the Czech Republic.A. baumannii was characterized from a surface water resources in South Nation River (SNR) drainage basin in Eastern Ontario, Canada, in 2013 [26].Besides, the composition of the bacterial community in a freshwater aquaculture environment was investigated in China by [29].The study reported that the relative abundance of Acinetobacter species was 0.5% of the total bacterial community.
All isolates that were positive for the recA (425 bp) (Figure S1) were taken as belonging to the Acinetobacter genus, according to [30].A PCR ampli cation assay based on recA gene-speci c primer, identi ed 844 Acinetobacter species of the 1107 presumptive isolates, while gyrB gene species-speci c primers further delineated them into species namely, A. baumannii (410) and A. nosocomialis (23).According to [30], the method used in this study was 98.2% speci c and 92.4% sensitive for Comparably, [31] demonstrated a PCR assay targeting recA gene A. baumannii, A. nosocomialis, and A. pittii are known nosocomial pathogens, which could cause multiple antibiotic resistant infections [2,30,32] in immune-compromised patients [2].Park and others [33] investigated the presence of Acinetobacter species in the bloodstream of patients with blood infection in a tertiary-care hospital in Korea between August 2003 and February 2010.Their ndings showed that A. baumannii and A. nosocomialis were prevalent in the samples collected.Similarly, [34], recovered 160 A. baumannii isolates from sputum, blood, pus and uid aspirates of patients in three Hospitals in Vietnam within a period of two years (between 2012 and 2014).These ndings showed that the nosocomial pathogens could colonize any part of the human body to cause infections.However, occurrence of A. baumannii and A. nosocomialis in an aquatic environment, is uncommon in the past few years.Nonetheless, in recent times, these nosocomial pathogens are isolated from other ecosystems [3,4,35].The isolation of the pathogen from water sources might be due to contamination coming from hospital wastewater and materials.
The virulence traits of A. baumannii and A. nosocomialis have been a major research focus in recent times [17,36].This is due to the nature of A. baumannii infections as well as the role virulence genes play in the pathogenicity of the emerging waterborne pathogen.Virulence genes are the mechanisms through which A. baumannii initiates pathogenesis [36,37], most especially in the clinical settings.
In this study, molecular assay for the detection of virulence factors in both pathogens was performed.
Figure S2 shows the electrophoretic images of the seven virulence factors evaluated.Although, A. baumannii had been con rmed to harbour several virulence factors (genes), seven of these genes (afa/draBC, epsA, mH, OmpA, PAI, sfa/focDE, and traT) are reported in this study.Reports on the virulence genes in A. baumannii are usually associated with isolates from clinical environment, whereas such reports are rare on A. baumannii from freshwater resources Thus, this study showed that OmpA gene was predominantly exhibited by the A. baumannii in all the rivers sampled, likewise mH and epsA genes were also detected in many of the isolates, whereas afa/draBC, PAI, Sfa/focDE, and traT genes were detected in a few Acinetobacter isolates.As such, the exhibition of virulence genes varies from one isolate to the other, which was also reported among clinical isolates known for nosocomial infections [38].The OmpA gene is the main outer membrane protein (OMP) located on the A. baumannii membrane [39,40], which [36].These observations corroborate the ndings of this current study which reports that the virulence pro les of an individual isolate varied greatly and OmpA gene was mostly detected [36].
Generally, the outer membranes of Gram-negative bacteria are made up of the OMPs, lipopolysaccharides and phospholipids layer [37].The presence of outer membrane protein A gene (OmpA) in the A. baumanni isolated from the freshwater source is of a major concern based on its role in pathogenesis [36].Besides, studies have shown that A. baumannii uses OmpA for adhesion to the lung epithelial cell by interacting with a cell cytoskeleton such as bronectin on the cell surface and thereby inducing pneumonia [41,42].It also causes cell death through caspase-3 activation [43,44].Similarly, A. baumannii could be responsible for apoptosis through the translocation of its OmpA into the mitochondria and the nucleus of host cells [45,46].The combination of the roles played by OmpA makes it an important virulence factor in the pathogenesis of A. baumannii infection.Moreover, antibiotic resistance in A. baumannii is also associated with OmpA [47,48].It was suggested that OmpA was involved in the removal of antibiotics from the periplasmic space membrane e ux systems [48].The survival and persistence of A. baumannii in the cell are enhanced by OmpA due to the formation of bio lms and surface motility.

Conclusions
In this study, three selected freshwater resources in the Eastern Cape Province, South Africa were evaluated for the occurrence, distribution and virulence genes ngerprints of clinically-relevant Acinetobacter species namely; A. baumannii and A. nosocomialis.This study suggests that these pathogenic Acinetobacter species could also inhabit aquatic environment.As a result, aquatic environment is an important reservoir for pathogenic Acinetobacter species as detected in this study.Besides, the OmpA is a major virulence factor associated with A. baumannii and A. nosocomialis in this study.As such, identi cation of these opportunistic and virulent waterborne pathogens in the freshwater resources requires public awareness and recognition as important to public health risks.

Description of study areas
Collection of water samples was carried out from three rivers namely; Great Fish, Keiskamma and Tyhume, in the Eastern Cape Province, South Africa between April 2017 and March 2018.The Great Fish River is located in Chris Hani District Municipality in the Eastern Cape Province and it is one of the major rivers used for irrigation and livestock farming in the area.This river is prone to agricultural and municipal runoffs and also serves as the receiving stream of e uents from many wastewater treatment plants (WWTPs), especially those situated in urban communities such as Craddock.Keiskamma and Tyhume rivers are located in the Amathole District Municipality in the Eastern Cape Province and are exposed to different anthropogenic activities from the rural and urban communities along the river courses, such as livestock drinking and irrigation farming.In addition, these rivers receive e uents from wastewater treatment plants (WWTPs) situated close to their banks.Different sampling points on these rivers were selected based on where humans and animals come into direct contact with them, for example, points where they are used for shing, drinking and swimming purposes, downstream of the WWTPs, points where irrigation water is released to the water bodies and proximity to hospital facilities.

Sampling
Water samples were collected at ve different sites (S1, S2, S3, S4, and S5) from Great Fish, Keiskamma and Tyhume rivers respectively, for a period of one year, which covers the four seasonal patterns in South Africa (autumn, winter, spring, and summer).Water samples were collected aseptically in sterile 1L glass bottles from different sampling points by midstream-dipping of sample bottles at 25-30 cm down the water column, with the mouth tilting against the ow of the river.All water samples were labelled properly and safely taken to the laboratory (in an ice chest) where they were processed within 6 h of collection [27].
Aliquots of water samples were used for isolation of Acinetobacter species based on standard microbiological procedures [49] Isolation and puri cation of presumptive Acinetobacter species The isolation of the presumptive density of Acinetobacter species in the water samples was determined by membrane ltration technique [50].Cellulose membrane of pore size 0.45 m was used to lter three volumes of 100 mL of the water samples under vacuum [50].These membranes were aseptically placed on plates with Acinetobacter species selective medium-CHROMagar Acinetobacter base plus selective supplement (CHROMagar, Paris, France) which was prepared according to the manufacturer' instruction.
Each sample plate was subjected to incubation at 37 o C for 24 h after inoculation.Each sample was analysed in triplicate.All bacterial colonies with red colouration on the CHROMagar plates were counted as presumptive Acinetobacter species and were expressed as CFU/100ml.All isolates were sub-cultured on nutrient agar using a streak plate method (Oxoid, UK) and puri ed for further species identi cation.Fifty percent (50%) glycerol stocks of the pure culture was prepared and stored at -80 ∘ C.

Molecular identi cation of Acinetobacter species by PCR assays
Extraction of genomic DNA: Presumptive Acinetobacter spp. in glycerol stocks was rst being resuscitated on tryptic soy broth and incubated for 18 to 24h at 37 ∘ C. DNA extraction from the bacterial isolates was carried out using the direct boiling method according to [51].The broth culture was centrifuged at 15000 rpm for 5 min using a Mini Spin Microcentrifuge (Lasec, RSA), then the supernatant was dispensed out and the pellet rinsed with sterile normal saline.The pellet was re-suspended in sterile distilled water and boiled in a heating block for 10 minutes using an AccuBlock (Digital dry bath, Labnet).

Delineation of Genus Acinetobacter into species
The con rmed Acinetobacter isolates were delineated into species accordingly [30,52,53] using a PCR assay.Firstly, optimization of conditions for A. baumannii, A. nosocomialis and A. pittii was carried out using species-speci c primers (Table S1 under supplementary Section), while reference strains DSM-102929, DSM-102856, and DSM-9341 (DSMZ, Germany) were used as positive controls for A. baumannii, A. nosocomialis and A. pittii respectively.The PCR ampli cation was performed as stated in the previous section.

Detection of virulence genes
Polymerase chain reactions were also carried out for the identi cation of some Acinetobacter virulence genes including afa/draBC, epsA, mH, OmpA, PAI, sfa/focDE, and traT genes, which have been previously found in clinical samples [54,55,56].The standard strain of A. baumannii DSM-30007 (DSMZ, Germany) was used as positive control.There was no positive control available for A. nosocomialis.The PCR assay and electrophoresis were conducted as earlier described.

Statistical Analysis
All statistical analyses were performed using the Statistica software v13.4.0.

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Table 2 :
14 (64-bits).A simple factorial ANOVA was performed for the comparison of normally distributed data.The p-values of less than 0.05 were considered statistically signi cant for all the statistical tests performed.Collection of freshwater samples for research purposes in this region does not require governmental permission since freshwater resources investigated are within the University jurisdiction.Also, the need for ethical approval was waived by the Research Ethics Committee, University of Fort Hare, because samples analysed in this study do not require human or animal subjects.Thummeepak R, Kongthai P, Leungtongkam U, Sitthisak S. Distribution of virulence genes involved in bio lm formation in multi-drug resistant Acinetobacter baumannii clinical isolates.International Microbiology.2016; 19:121-129 doi:10.2436/20.1501.01.270.55.Momtaz H, Seifati SM, Tavakol M. Determining the prevalence and detection of the most prevalent virulence genes in Acinetobacter baumannii isolated from hospital infections.Int J Med Lab.2015; 2(2):87-97.5 .Braun G, Vidotto MC.Evaluation of Adherence, Hemagglutination, and Presence of Genes Codifying for Virulence Factors of Acinetobacter baumannii Causing Urinary Tract Infection.Mem Inst Oswaldo Cruz, Rio de Janeiro, 2004; 99(8): 839-844.Tables Table Summary of the relative abundance of the genus Acinetobacter and two clinically important species the freshwater studied Virulence factors of A.baumannii and A. nosocomialis isolates in addition to which river they belong to.