Multidrug-resistant Vibrio species isolated from abattoir effluents in Nigeria

Introduction: The antibiograms of Vibrio species isolated from abattoir effluents in the Niger Delta region of Nigeria were investigated with respect to their public health significance. Methodology: Vibrio species were isolated and identified using standard microbiological and molecular techniques, while antibiogram of isolates was tested and interpreted using the disk diffusion method described by the Clinical and Laboratory Standards Institute. Results: Of 150 presumptive isolates, 48 (32%) were confirmed to be Vibrio spp. by PCR; of these, 23 (47.9%) were V. cholerae, 11 (22.9%) were V. fluvialis, 8 (16.7%) were V. vulnificus, and 6 (12.5%) were V. parahaemolyticus. The antibiogram revealed that Vibrio species were generally resistant to ampicillin (60%–67%), trimethoprim (80%–100%), and tetracycline (60%–83%), whereas they were sensitive to ceftriaxone (86%–100%), the aminoglycosides (67%–100%), imipenem (86%–100%), ofloxacin (83%–100%), and chloramphenicol (67%– 100%). The isolates exhibited multiple antibiotic resistance (MAR) with an average MAR index of 0.23. Conclusions: This study demonstrated that abattoir effluents are important reservoirs for multidrug-resistant Vibrio species that might be considerable contributors to the recurrent episodes of epidemic cholera and non-Vibrio cholera infections in Nigeria.


Introduction
An abattoir is a special facility designed and licensed for receiving, holding, slaughtering, and inspecting meat animals and meat products before release to the public [1].Abattoir inspection of live animals (ante-mortem) and carcasses (post-mortem) is critical to surveillance for animal diseases and zoonoses [2].Cadmus et al. [3] reported that pathogens of zoonotic importance are associated with more than 80% of public abattoirs in Nigeria.This observation has serious public health implications, as many Nigerian abattoirs dispose their effluents directly into streams and rivers without any form of treatment [4].Incidentally, these streams and rivers also serve as water resources for domestic, agricultural, recreational, as well as drinking purposes for communities and settlements downstream.It is little wonder, therefore, that waterborne diseases such as cholera are recurring in Nigeria.
Antibiotics are often employed as feed additives to promote rapid growth of livestock [5], thereby contributing to increased incidence of antibiotic resistance among bacterial species that inhabit abattoir effluents, due to selective pressure [1].Emergence of microbial resistance to multiple drugs is an ongoing challenge that threatens the effectiveness of antibiotics in the continuous management of infectious diseases, especially in low-and medium-income countries (many of which are in Africa) lacking relevant infrastructure and institutions targeted at making sanitation and water resources available and accessible to all.A good example is a work from Guinea Bissau that reported that multiple-antibiotic resistance was responsible for the increase in fatality from 1% to 5.3% during a cholera outbreak that occurred between 1996 and 1997 [6].
Although abattoir effluents have been reported [7,8] to be important environmental reservoirs for Vibrio species, there is a dearth of information in the literature on antibiotic susceptibility patterns of Vibrio species isolated from abattoir effluents in Nigeria.Therefore, the aim of this study was to investigate the antibiogram of Vibrio species isolated from abattoir effluents in the Niger Delta region of Nigeria.

Isolation and preliminary identification of Vibrio species
Aliquots of the samples were inoculated into alkaline peptone water (APW, Pronadisa, Madrid, Spain) and incubated aerobically at 37°C for 18 to 24 hours.Turbid cultures were streaked onto thiosulphate citrate bile salts sucrose (TCBS) agar (Pronadisa, Madrid, Spain) and incubated at 37°C for 24 hours.Suspected Vibrio species appear as green or yellow colonies on TCBS.Five to ten isolated colonies per plate were randomly picked from each sample and subcultured onto fresh TCBS agar plates.The pure isolates were subjected to preliminary identification using standard cultural and biochemical methods as described by Kaysner and DePaola [9].The identity of presumptive Vibrio isolates were further confirmed using the polymerase chain reaction (PCR) technique described below.

Molecular confirmation of Vibrio isolates
Isolates identified as Vibrio species by cultural/biochemical techniques were confirmed by PCR using the specific primers described in Table 1.DNA extraction and PCR were carried out as described by Igbinosa et al. [10], with slight modifications.Single colonies of presumptive Vibrio strains grown overnight at 37°C on TSA-2% NaCl agar plates were picked, suspended in 200 µL of sterile Milli-Q PCR grade water (Merck, Modderfontein, South Africa), and the cells lysed using Dri-block DB.2A (Techne, Cape Town, South Africa) for 15 minutes at 100°C.The cell debris were removed by centrifugation at 11,000 × g for 2 minutes using a MiniSpin micro centrifuge (Merck, Modderfontein, South Africa).The cell lysates (10 µL) were used as a DNA template in the PCR assays immediately after extraction or following storage at -20°C.Sterile Milli-Q PCR-grade water (Inqaba Biotec, Pretoria, South Africa) was included in each PCR assay as a negative control.The thermal cycling condition was as follows: initial denaturation at 93°C for 15 minutes, followed by 35 cycles of denaturation at 92°C for 40 seconds, annealing at 57°C for 1 minute and extension at 72°C for 1.5 minute, and a final extension step of 72°C for 7 minutes.The amplified products were held at 4°C after completion of the cycles prior to electrophoresis.For V. fluvialis, the amplification condition was as follows: initial denaturation at 94°C for 5 minutes, followed by 30 cycles consisting of denaturation at 94°C for 40 seconds, annealing at 65°C for 40 seconds, and extension at 72°C for 1 minute.The PCR products were electrophoresed in 1.5% agarose gel containing 0.5 mg/L ethidium bromide for 40 minutes at 100 V and then visualized using a UV transilluminator.

Detection of cholera toxin gene (ctx)
The extraction of genomic DNA from Vibrio isolates was as described above.The protocol described by Kaysner and DePaola [9] was used for detection of the ctx toxigenic gene in suspected strains of V. cholerae.The primer set used was 5'-TGA AAT AAA GCA GTC AGG TG-3' (forward) and 5'-GGT ATT CTG CAC ACA AAT CAG-3' (reverse), and the size of Source: Igbinosa et al. [11] the expected PCR amplicon was 777 bp.The amplification reaction consisted of an initial denaturation step of 94°C for 3 minutes and 35 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C, with a final extension step of 3 minutes at 72°C.

Antibiotic susceptibility test
Susceptibility of Vibrio isolates to antimicrobial agents was performed using the disk diffusion method following guidelines established by the Clinical and Laboratory Standards Institute [11]

Multiple antibiotic resistance (MAR) index
MAR index was calculated as previously described by Blasco et al. [12] as MAR = a/b, where a is the number of antibiotics to which an isolate was resistant and b is the total number of antibiotics against which individual isolates were tested.
MAR index higher than 0.2 identifies organisms that originate from high-risk sources of contamination where antibiotics are often used or abused [13].
Table 3 shows the resistance pattern of the test isolates assessed in this study.Two strains of V. vulnificus were completely susceptible to all the test antibiotics deployed in this study and hence had no resistance record.The other isolates exhibited MAR in combinations ranging from 2 to 12 antibiotics, except in strains of V. parahaemolyticus and V. vulnificus that exhibited mono-resistance to ceftazidime and tetracycline, respectively (Table 3).The MAR index ranged between 0 and 0.63; the highest MAR index was observed in a strain of V. cholerae isolated from Oghara, while the lowest was expectedly observed in the two strains of V. vulnificus that were completely sensitive to all the tested antibiotics.

Table 1 .
List of primers used in this study.

Table 2 .
Antibiotic susceptibility profiles of the Vibrio strains isolated from abattoir effluents. .V. vulnificus were generally sensitive to the test antibiotics, with a few showing low resistance to ceftazidime and trimethoprim, among others (Table * Trimethoprim/sulphamethoxazole; S: sensitive; I: intermediate; R: resistant.sulfamethoxazole