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Open Access Journal of Microbiology & Biotechnology Research Article 9 min read

Antimicrobial Resistance Profile Characterization of Enterococcus Species Isolated from Aquaculture Environment

Igbinosa EO, Onuoha T and Udochukwu U*
* Corresponding author
ISSN: 2576-7771  10.23880/oajmb-16000107  Received: September 08, 2016  Published: September 28, 2016
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Keywords
Antimicrobial Resistance Profile Enterococcus Species Aquaculture Environment Antibiotics
Abstract

Aquaculture environments can become reservoirs of antibiotic-resistant strains of faecal bacteria including enterococci   which are capable of contributing to the spread of antimicrobial resistance in the marine environment. The aim of this   study was to characterize the antimicrobial resistance profile of Enterococcus species from various aquacultures (fish   ponds) in Benin City, Edo State. A total of 376 water samples were obtained from four different fish ponds between   February and July, 2015 and were investigated for the presence of Enterococcus species using standard culture based   methods. The antimicrobial susceptibility profiles of the isolates were determined by using Kirby-Bauer disc diffusion   method. From the 376 water samples analyzed for the presence of enterococci isolates, 100 isolates each from   Enterococcus faecalis and Enterococcus faecium were isolated from the four fish ponds investigated. The mean   enterococal counts from the four fish ponds investigated ranged from 4±0.01 × 102 to 12±0.17×102 cfu/ml. Statistical   analysis reveals that there was a significant difference observed in the mean enterococcal counts obtained from the four   different fish ponds in the study (p< 0.05). The distribution of the enterococcal isolates in the study from February to July   reveals that 50 isolates from each pond were characterized. Statistical analysis also reveals that there was a significant   difference observed in the distribution of the enterococcal isolates from the fish ponds (p< 0.05). High level resistance   was observed against six antibiotics used in the study such that 15 enterococcal isolates displayed marked resistance to   the action of trimethoprim, chloramphenicol, erythromycin, oxytetracycline, sulfamethoxazole, and ciprofloxacin.   Adherence to adequate and proper use of manure products and frequent discharge of water from fish pond will reduce   the high level of antimicrobial resistance in Enterococcus species isolated from fish ponds and also reduce the potential   risk to human health.  

Introduction

The rapid emergence and dissemination of antibiotic- resistant microorganisms in the global ecosystem has been widely reported as a significant public health issue, thus there is concern on the future ability to treat multi- drug resistant infections. Contaminated ponds can become reservoirs of virulent and antibiotic-resistant strains of faecal bacteria [1], including enterococci [2], which are capable of transmitting resistance genes to other bacteria by horizontal gene transfer mechanisms, thus contributing to the spread of resistance genes into the marine environment. Aquaculture has been a growing activity for the last 30 years worldwide and has become one of the fastest developing sources of animal protein to humans and animals due to dwindling wild fish stocks around the world, in particular Nigeria [3]. The importance of fish cannot be overemphasized. Fish is a low fat food, a great source of protein, vitamins and minerals. Annual domestic fish supply in Nigeria stands at about 400,000 tons [4]. Accumulation of surplus antimicrobials and antimicrobial residues may occur when the ponds are only rarely emptied at the time of fish harvest. Such a build-up could establish selective pressure favoring selection and growth of antimicrobial-resistant bacteria [5]. Enterococci are Gram positive cocci that are part of the human and animal intestinal microflora and are used as faecal indicator bacteria for assessing potential risks for human health and monitoring recreational waters [6]. Due to the ability of Enterococci to transfer transposons (including conjugative transposons), resistance plasmids, and sex pheromone plasmids to a broad range of recipients. Effective water management in fish ponds is one of the important factors that contribute to the success of fish culture [7].

Materials and Methods

Sample Collection

The water samples were obtained from three different fish ponds (P1, P2 and P3) where there was a deliberate impute of 100mg/kg animal waste/manure, while samples were also obtained from a fish pond (P4) where there was no history of animal waste/manure application. The sampling period was between February and July, 2015 in Benin City, Edo State. A total of 376 water samples were obtained from the four different fish ponds investigated in the study. The size of the ponds varies between 0.8 and 1.2 hectare. The fish ponds were located in Ikpoba hill, Sapkonba and Iyekeogba Estate, Benin City. During each visit to the pond, water discharge sample were collected from the fish pond by using clean plastic syringes and tube and were transported to the laboratory for microbiological analysis in cold ice pack and analyzed within 24h after collection [1].

Isolation of Bacterial Isolates from Water Samples

All media used for this study were prepared according to the manufacturer’s instruction. The media used in the study include Bile Esculin agar, Nutrient agar, and Mueller Hilton agar. Isolation of Enterococcus species was carried out as described by [8]. One millilitre (1 ml) of the water samples was transferred into a sterile test tube containing 9.0 ml of peptone broth. This process was repeated for other sterilized test tubes so that at the end, dilution of 10-1, 10-2 and 10-3 dilutions were obtained. A 0.1 ml from 10-1, 10-2 and 10-3 dilution was then plated out using the pour plate method on Bile Esculin agar plates [9]. The Bile Esculin agar plates were incubated at 37°C for 24 - 48 h, after incubation the colonies were counted and expressed in colony forming unit per millilitre (cfu/ml). Discrete colony were sub-cultured on freshly prepared Bile Esculin agar plates, incubated at 37°C for 24-48 h and stored on Nutrient agar slant at 4°C for further laboratory analysis.

Characterization and Identification of Bacterial Isolates

The total viable bacteria counts (TVBC) formed were counted using a colony counter. Characterization of isolates was carried out as described by [8]. Bacterial isolates were identified on the basis of cultural, morphological, biochemical and sugar fermentation tests such as glucose, lactose, arabinose, sucrose, maltose, and mannitol [10].

Antibiotic Susceptibility Screening

The enterococcal isolates that were positively identified using the culture based methods were subjected to antibiogram characterization [11] All the bacterial isolates were tested for resistance or sensitivity to different antibiotics using the standard disc diffusion method (Kirby Bauer test). For the disc diffusion assay, bacteria were grown between 18 and 24 h on Mueller- Hinton agar, harvested and then suspended in 0.85% sterile physiological saline solution adjusted to a 0.5 McFarland turbidity standard, corresponding to 108 cfu/ml. The inoculum was streaked on plates of Mueller- Hinton agar using a sterile cotton swab and impregnated with appropriate antibiotics [12]. The results were recorded after 24 h of incubation at 37°C. Commercially available antibiotics discs, obtained from Mast

Diagnostics, Merseyside, United Kingdom, were used to determine the resistance patterns of the isolates against 6 different antibiotics [13]. The diameter of the zone of inhibition around each disc was measured and interpreted as Resistant (R), Intermediate resistant (I) or Sensitive (S) in accordance with the recommended standard established by the Clinical Laboratory Standards Institute [14]. All data in the study were analyzed using the statistical package (SPSS). P-values < 0.05 were considered statistically significant.

Results and Discussions

MonthsBacterial isolates (102 cfu/ml)P-value
Pond 1Pond 2Pond 3Pond 4
February10±0.01B12±0.02A14±0.03B12±0.00A0.014
March16±0.00B12±0.05A20±0.02C18±0.02BC0.032
April18±0.31B20±0.01C18±0.00B16±0.00A0.002
May20±0.00B22±0.00C22±0.00C14±0.01A0.015
June20±0.12C18±0.02B14±0.01A26±0.13D0.032
July22±0.17A26±0.20C22±0.04A24±0.01B0.001

Table 1: Mean enterococcal counts from the various ponds.

of the enterococcal isolates were resistant to the action of chloramphenicol; 39/200 (19.50%) of the enterococcal isolates were resistant to the action of erythromycin; 15/200 (7.50%) of the enterococcal l isolates were resistant to the action of oxytetracycline; 9/200 (4.50%) of the enterococcal isolates were resistant to the action of sulfamethoxazole; 89/200 (44.50%) of the enterococcal l isolates were resistant to the action of ciprofloxacin. Statistical analysis reveals that there was no significant difference observed in the antimicrobial resistance profile of the enterococcal isolates from the fish ponds (P> 0.05).

  • Multidrug resistance profile
  • Number of isolates n=200
  • SMXR, OXYR
  • 89
  • SMXR, OXYR, CHLR
  • 39
  • SMXR, OXYR, CHLR, TRIR
  • 25
  • SMXR, OXYR, CHLR, TRIR, ERYR
  • 21
  • SMXR, OXYR, CHLR, TRIR, ERYR, CIPR
  • 15

Table 2: Multidrug resistance profile of the Enterococcal

TRI: Trimethoprim; CHL: Chloramphenicol; ERY: Erythromycin; OXY: Oxytetracycline; SMX: Sulfamethoxazole; CIP: Ciprofloxacin.

Pond 1Pond 2Pond 3Pond 4
SamplingTotalP-
PeriodE.E.E.E.E.E.E.E.n=200value
faecalisfaeciumfaecalisfaeciumfaecalisfaeciumfaecalisfaecium
n=32n=18n=20n=30n=12n=38n=40n=10
4
(12.50)		3
(16.67)		3
(15.00)		5
(16.67)		3
(25.00)		7
(18.42)		6
(15.00)		2
(20.00)		33
(16.50)		0
February
3 (9.38)5
(27.78)		4
(20.00)		6
(20.00)		2
(16.67)		5
(13.16)		7
(17.50)		1
(10.00)		33
(16.50)		0.001
March
6
(18.75)		2
(11.11)		3
(15.00)		5
(16.67)		2
(16.67)		3 (7.90)8
(20.00)		1
(10.00)		30
(15.00)		0.003
April
5
(15.63)		3
(16.67)		2
(10.00)		4
(13.33)		2
(16.67)		4
(10.53)		5
(12.50)		2
(20.00)		27
(13.50)		0
May
6
(18.75)		3
(16.67)		5
(25.00)		3
(10.00)		1 (8.33)9
(23.68)		6
(15.00)		1
(10.00)		34
(17.00)		0.002
June
8
(25.00)		2
(11.11)		3
(15.00)		7
(23.33)		2
(16.67)		10
(26.32)		8
(20.00)		3
(30.00)		43
(21.50)		0.002
July

Table 3: Distribution of the enterococcal isolates from the fish ponds Values in parenthesis represents (%)

Pond 1Pond 2Pond 3Pond 4
E.E.E.E.E.E.E.E.TotalP-
Antibiotics
faecalisfaeciumfaecalisfaeciumfaecalisfaeciumfaecalisfaeciumn=200value
n=32n=18n=20n=30n=12n=38n=40n=10
20
(62.50)		1 (5.56)0 (0)2 (6.67)0 (0)2 (5.26)0 (0)0 (0)25
(12.50)		0.239
TRI
1 (3.13)15
(83.33)		0 (0)2 (6.67)1 (8.33)1 (2.63)0 (0)1
(10.00)		21
(10.50)		0.184
CHL
1 (3.13)0 (0)17
(85.00)		20
(66.67)		1 (8.33)0 (0)0 (0)0 (0)39
(19.50)		0.147
ERY
0 (0)1 (5.56)1 (5.00)4 (13.33)0 (0)0 (0)10 (25)1
(10.00)		15
(7.50)		0.124
OXY
0 (0)1 (5.56)0 (0)1 (33.33)2
(16.67)		0 (0)5
(12.50)		0 (0)9
(4.50)		0.108
SMX
10
(31.25)		0 (0)2
(10.00)		1 (33.33)8
(66.67)		35
(92.11)		25
(62.50)		8
(80.00)		89
(44.50)		0.04
CIP

Table 4: Antimicrobial resistance profile of the enterococcal isolates from the fish ponds.

Values in parenthesis represents (%); TRI: Trimethoprim; CHL: Chloramphenicol; ERY: Erythromycin; OXY: Oxytetracycline; SMX: Sulfamethoxazole; CIP: Ciprofloxacin.

Environmental water quality studies may benefit however from focusing on a subset of Enterococcus spp. that are associated consistently with sources of faecal pollution such as domestic sewage, rather than testing for presence of the entire genus. E. faecium and E. faecalis are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage [15]. In this study, the presence of Enterococcus species (E. faecalis and E. faecium) from fish ponds in Benin City, Edo State has been demonstrated. Reports from this investigation shows a high counts of Enterococcus species ranging from 2±0.00×102 to 16±0.13×102 cfu/ml. The findings in the study were within range when compared to the findings of [16] were 1.2×102 to 1.8×103 cfu/ml isolated from fish ponds in Kano metropolis. Enterococci which have been implicated with warm-blooded animals, their faeces, animal carcasses or milk, are also able to colonise a diversity of niches, mainly because of their exceptional aptitude and intrinsic resistance against hostile conditions [6]. Previous studies have demonstrated that enterococci have a strong relationship with swimming associated illnesses in both marine and fresh water environments [15]. The public health costs associated with exposure to faecal contamination may have a huge economic impact. A total of 104/200 (52%) E. faecalis and 96/200 (48%) E. faecium were isolated from the fish ponds in the study. Multidrug resistance profile of the isolates in the study reveals that 15 of the isolates were resistant to all antimicrobials used in the study [17]. Overuse and misuse of antimicrobials in food animals represent a public health risk as they contribute to the emergence of resistant forms of disease-causing bacteria which is a contributing factor to their pathogenesis [18].

Conclusion

The information obtained from the study reveals that Enterococcus faecalis and Enterococcus faecium were highly resistant to the action of antimicrobials used in the study, in Benin City, Edo State. These antimicrobials are usually used as prophylactics, growth promoters, and also as treatment to the fishes in the fish ponds. Resistance to these antimicrobials could be attributed to selective pressure, possession of intrinsic and acquired resistance to the antimicrobials in the fish ponds and fish pond facilities in Benin City. Adherence to adequate and proper use of manure products and frequent discharge of water from fish pond will reduce the high level of antimicrobial resistance in Enterococcus species isolated from fish ponds and also reduce the potential risk to human health.

References

  1. Bates J, Jordens JZ, Griffith DT (1994) Farm animals as a putative reservoir for vancomycin resistant enterococcal infections in man. J Antimicrob Chemother 34(4): 507-514.
  2. Bager F, Madsen M, Christensen J, Aarestrup FM (1997) Avoparcin used as a growth promoter is associated with the occurrence of vancomycinresistant Enterococcus faecium in Danish poultry and pig farms. Prev Vet Med 31(1-2)**:** 95-112.
  3. McPhearson RM, DePaola A, Zywno SR, Motes ML, Guarino AM (1991) Antibiotic resistance in Gram- negative bacteria from cultured catfish and aquaculture ponds. Aquaculture 99(3-4)**:** 203-211.
  4. Alderman DJ, Hastings TS (1998) Antibiotic use in aquaculture development of antibiotic resistance potential for consumer health risks. International Journal of Food Science and Technology 33(2)**:** 139- 155.
  5. Aarestrup FM (1995) Occurrence of glycopeptide resistance among Enterococcus faecium isolates from ecological and conventional poultry farms. Microb Drug Resist 1(3): 255-257.
  6. Guardabassi L, Dalsgaard A, Raffatellu M, Olsen JE (2000) Increase in the prevalence of oxolinic acid resistant Enterococcus spp. observed in a stream receiving the effluent from a freshwater trout farm following the treatment with oxolinic acid-medicated feed. Aquaculture 188(3-4)**:** 205-218.
  7. Schmidt AS, Bruun MS, Dalsgaard I, Pedersen K, Larsen JL (2000) Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four Danish rainbow trout farms. Applied Environmental Microbiology 66(11)**:** 4908-4915.
  8. McFaddin JF (1985) Media for isolation-cultivation- identification-maintenance of medical bacteria. Williams & Wilkins, USA.
  9. Anderson NL. Cumitech 3B: Quality Systems in the Clinical Microbiology Laboratory. ASM Press, Washington DC.
  10. Jensen BB (1998) The impact of feed additives on the microbial ecology of young pigs. Journal of Animal and Feed Sciences 7: 45-64.
  11. Leclerq R, Derlot E, Duval J, Courvalin P (1988) Plasmid mediated resistance to vancomycin and eicoplanin in Enterococccus faecium. N Engl J Med 319: 157-161.
  12. Klare I, Heier H, Claus H, Reissbrodt R, Witte W (1995) VanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry. FEMS Microbiol Lett 125(2-3): 165-172.
  13. Wegener HC (1998) Historical Yearly Usage of Glycopeptides for animals and humans: the American-European paradox revisited. Antimicrob Agents Chemother 42(11): 3049.
  14. CLIS (2006) Performance standards for antimicrobial susceptibility testing: Eleventh informational supplement. CLIS, Wayne, Pa.
  15. Laukova A, Juris P (1997) Distribution and characterization of Enterococcus species in municipal sewages. Microbios 89(359)**:** 73-80.
  16. McDonald CL, Kuehnert MJ, Tenover FC, Jarvis WR (1997) Vancomycin-resistant enterococci outside the health care setting: prevalence, sources, and public health. Emerg Infect Dis 3(3): 311-317.
  17. Singh N, Rogers P, Atwood CW, Wagener MM, Yu VL (2000) Short-course empiric antibiotic therapy for patients with pulmonary infiltrates in the intensive care Unit: A proposed solution for indiscriminate antibiotic prescription. Am J Respir Crit Care Med 162(2): 505-511.
  18. Witte W (1998) Medical consequences of antibiotic use in agriculture. Science 279(5353): 996-997.
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@article{igbinosa2016,
  title   = {Antimicrobial Resistance Profile Characterization of
Enterococcus Species Isolated from Aquaculture
Environment},
  author  = {Igbinosa EO, Onuoha T and Udochukwu U},
  journal = {Open Access Journal of Microbiology & Biotechnology},
  year    = {2016},
  volume  = {1},
  number  = {2},
  doi     = {10.23880/oajmb-16000107}
}
Igbinosa EO, Onuoha T and Udochukwu U (2016). Antimicrobial Resistance Profile Characterization of
Enterococcus Species Isolated from Aquaculture
Environment. Open Access Journal of Microbiology & Biotechnology, 1(2). https://doi.org/10.23880/oajmb-16000107
TY  - JOUR
TI  - Antimicrobial Resistance Profile Characterization of
Enterococcus Species Isolated from Aquaculture
Environment
AU  - Igbinosa EO, Onuoha T and Udochukwu U
JO  - Open Access Journal of Microbiology & Biotechnology
PY  - 2016
VL  - 1
IS  - 2
DO  - 10.23880/oajmb-16000107
ER  -