Antimicrobial Resistance of Shigatoxin Producing Escherichia coli 0157:NM Isolates from Water Fed to Cattle in Northwestern Nigeria
Shiga toxin-producing Escherichia coli 0157:NM (STEC 0157:NM) was isolated from water fed to cattle and its resistance to antimicrobial agents was determined. Five hundred water samples were collected from cattle establishments in northwestern Nigeria from December 2002 to June 2006. Serological confirmation and Shiga toxin production were carried out using kits (Oxoid, Basingstoke England). Resistance to 16 antimicrobials was tested by the standardized disc deffusion method. STEC 0157:NM was isolated from 0.4% (2/500) of the samples. The isolates were resistant to several commonly used antimicrobial agents in livestock health and production. There was no resistance to chloramphenicol and fluoroquinolones but quinolone resistance was observed. The emergence of STEC 0157:NM in water used for feeding cattle in northwestern Nigeria may pose a serious health hazard to livestock and humans. Legislation on import control of livestock, veterinary drug acquisition and use and public education on drug use and its implications are essential to manage this threat to public health and the livestock industry.
to cite this article:
I.I. Luga, I. Akodu, L.I. Mhomga, L. Allam, I. Ajogi, V.J. Umoh and J.K.P Kwaga, 2007. Antimicrobial Resistance of Shigatoxin Producing Escherichia coli 0157:NM Isolates from Water Fed to Cattle in Northwestern Nigeria . Asian Journal of Animal and Veterinary Advances, 2: 205-211.
Escherichia coli normally reside in the gastrointestinal tract of man and animals. It is a member of the coliform group of bacteria and some serotypes are leading causes of diarrhea in man and animals (Bertolini et al., 2006; Rao et al., 2006).
Shiga toxin-producing Escherichia coli (STEC) are an emerging group of pathogens of public health significance on a global scale (Allison et al., 2000; Armstrong et al., 1996; FSAI, 1999; Fukushima et al., 1999; Germani et al., 1998; Riley et al., 1983). Escherichia coli 0157:NM is the most common STEC in Africa (Effler et al., 2001). This pathogen contaminates water sources and has been shown to cause death in man and animals. In the outbreak in South Africa and Swaziland, 40,912 human diarrhea cases and a seven-fold increase in cattle death during the period of the outbreak was reported (Effler et al., 2001).
Therefore, this study was design to isolate E. coli 0157:NM in water fed to cattle, determine its Shiga toxin production profile and resistance to some commonly used antimicrobial agents in northwestern Nigeria.
MATERIALS AND METHODS
The multistage sampling method (Snedecor and Cochran, 1976) was employed to
select Kaduna and Sokoto States from seven states in the northwestern geo-political
zone of Nigeria. Four out of the six dairy farms and two of the four dairy farms
in Kaduna and Sokoto States, respectively were selected as sampling sites. Supplementary
water samples were collected randomly from beef cattle farms and nomadic Fulani
cattle herds around Zaria, Kaduna State and Sokoto municipal areas from December
2002 to June 2006.
The water was sampled by collecting 100 mL of untreated and 200 mL of chlorinated tap water. The samples were collected using sterile equipment and procedures and stored in cool boxes (Ayres and Mara, 1996).
Water samples were assayed for E. coli 0157:NM using the membrane
filtration technique (Ayres and Mara, 1996), at the Departments of Veterinary
Public Health and Preventive Medicine, Ahmadu Bello University, Zaria and Usmanu
Danfodiyo University, Sokoto. Filtration (pore size of 0.45ìm) was followed
by enrichment in modified tryplone soya broth (MTSB) supplemented with novobiocin
(Oxoid Ltd., Basingstoke, England) and incubation at 37±1°C for 24
h. The enriched filters were then cultured on Cefixime-Sorbitol-MacConkey (CR-SMAC)
agar (Oxoid Ltd., Basingstoke, England) and incubated for 24 h at 37±1°C.
Colourless colonies were selected and stored at 4°C on nutrient agar (NA)
Biochemical screening was carried out as recommended by Cowan et al.
(1981). This included testing for citrate utilization; urease production; indole,
sulphide production and motility using sulphide indole and motility (SIM) medium;
Hydrogen sulphide (H2S) production, fermentation of glucose, sucrose
and/or lactose and gas production using Triple Sugar Iron (TSI) agar; methyl
red and Voges-Proskauer tests using methyl red and Voges-Proskauer (MRVP) medium;
reaction from fermentation of lactose, maltose, manitol, arabinose and sorbitol
and gas from glucose fermentation.
The remel Wellcolex E. coli 0157:H7 kit (Remel Europe Ltd. Kent UK)
was used. It is a rapid latex agglutination test for the indentification of
the somatic (0157) antigen and flagellum (H7) antigen with specific antibodies.
The test was controlled by using a reference strain of E. coli 0157:H7
Detection of Shiga Toxin Production
The production of Shiga toxin 1 (ST1) and/or Shiga toxin 2 (ST2) was detected
using VTEC-RPLA kit (Oxoid Ltd., Basingstoke, England). Polymyxin B (Oxoid Ltd.
Basingstoke England) at a concentration of 5000 units mL was used to facilitate
the release of the Shiga toxins from the isolates (Karmali et al., 1985)
and the solid culture method was carried out. Serologically confirmed isolates
of E. coli 0157:NM were inoculated onto Brain Heart Infusion (BHI) agar
slopes (10 mL) and incubated at 37±1°C for 20 h. The growth was suspended
in 1 mL of 0.85% saline containing 5000 units of Bacillus cereus selective
supplement (Polymixin B) (Oxoid Ltd., Basingstoke, England). Extraction was
carried out for 30 min at 37°C in a vibrating water bath. Each culture extract
was cooled at 4°C for 30 min in a refrigerator and them centrifuged at 4000
rpm for 20 min.
The extract was used to assay for Shigatoxins following the manufacturers instructions to the letter and reading agglutination in the v-well microtitre plates.
Identification of STEC 0157:NM
Isolates that were citrate negative; urease negative; indole positive, H2S
negative, non-motile using SIM medium; H2S negative, produced an
acid/acid reaction with gas production on TSI agar; methyl red positive, Voges-Proskauer
negative using MRVP medium; produced acid reaction from fermentation of lactose,
maltose, manitol, arabinose and sorbitol; produced gas from glucose fermentation;
produced agglutination of 0157 test latex accompanied by a lack of agglutination
of the control latex; lack of agglutination of the H7 test latex accompanied
by a lack of agglutination of the H7 control latex; agglutination of test sample
supernatant and test latex (VT1 and/or VT2), lack of agglutination in the wells
containing latex control and no samples respectively, agglutination of verotoxin
1 and 2 controls - assayed as the test latex above; were considered to be STEC
Antimicrobial Resistance Testing
In vitro resistance tests were performed by the standardized disc
diffusion method on Mueller-Hinton agar (MHA) (Bauer et al., 1966). From
a pure culture of E. coli 0157:NM on SMAC agar, 5 colourless colonies
of the organism were transferred with a wire loop to a test tube containing
4mL of Tryptone Soya Broth (TSB). The tubes were incubated at 37°C for 5
h. The resulting suspension was standardized by diluting with 0.85% saline to
a density visually equivalent to the McFarland Standard of 0.5 (a turbidity
standard prepared by adding 0.5 mL of 1% barium chloride solution to 99.5 mL
of 1% H2SO4). This equated to approximately 108
of E. coli 0157:NM per mL of the suspension.
MHA plates were prepared and dried in an incubator. Cotton swabs were immersed into the suspension and the entire surface of the MHA was spread evenly with the suspension. The inoculum was allowed to dry with the plates covered. Using aseptic technique each disc was placed and pressed to ensure full contact with the agar. The inverted MHA plates were immediately incubated at 37+1°C for 14 h. Each disc set was validated by using the control strain-ATTC 43895.
Zones of inhibitions around the disc were measured to the nearest millimeter.A disc set was considered valid if it produced zones of inhibition ≥15 mm on MHA agar using ATTC 43895. A strain was considered stable (resistant) if the diameter of the zone was ≤10 mm, moderately sensitive being 11-14 mm and sensitive ≥15 mm. Zones of ≥26 mm indicated high sensitivity (Krivoshein et al., 1989). A total of 16 antimicrobial disc (Oxoid Ltd. Basingstoke England), were used for the tests. The antimicrobials tested were the following: ampicillin, cephazolin, gentamincin, kanamycin, neomycin, streptomycin, tetracycline, sulphamethoxazole, compound sulphonamides, co-trimoxazole, ceftazidime, cefuroxime, chloramphenicol, ciprofloxacin, nalidixic acid and norfloxacin (Shroeder et al., 2002; Ramon-Yusuf et al., 1990).
STEC 0157:NM Isolation
Shiga toxin-production was detected in 2 isolates making the isolation rate
of Shiga toxin-producing E. coli 0157:NM to be 0.4% (2/500). Only Stx2
was detected from the two isolates (Table 1).
Antimicrobial Resistance among STEC 0157:NM Isolates
The two isolates were resistant to several antimicrobials including ampicilin,
streptomycin, sulphamethoxazole, compound sulphonamides, tetracycline and nalidixic
acid. One isolate was resistant to neomycin and co-trimoxazole. There was no
resistance to chloramphenicol, kanamicin, gentamycin, ceftazidime, cefuroxime,
cephazolin, norfloxacin and ciprofloxacin. High sensitivity was observed to
chloramphenicol, norfloxacin and ciprofloxacin (Table 2).
Antibiograms of STEC 0157:NM from Water
There were two antimicrobial resistance patterns. Ampicillin-Streptomycin-Sulphamethoxazole
-Compound Sulphonamides-Tetracycline-Nalidixic acid and Ampicillin-Neomycin-Streptomycin-Sulphamethoxazole-Co-trimoxazole-Compound
Sulphonamide-Tetracycline-Nalidixic acid (Table 3).
||Shigatoxin-production in E. coli 0157: NM isolates from water
|The figures in parenthesis are percentages; NT, Number tested;
NN, CR-SMAC Negative; NP, number positive
|| Antimicrobial resistance among two STEC 0157:NM isolates from water
|1Isolates from a dairy farm watering points; *High
|| Patterns of antimicrobial resistance of 2 isolates of STEC 0157: NM from
a dairy farm watering point
|aAMP, ampicillin; S: Streptomycin; RL: sulphamethoxazole;
S3: compound s ulphonamides; TE: tetracycline; NA: nalidixic acid; N: neomycin
and SXT: suphamethoxazole/trimethoprim (co-trimoxazole)
The isolation of STEC 0157:NM should be of interest and concern to the public,
dairy and fast food industries in Nigeria because this pathogen causes bloody
diarrhea and death in man and animals respectively. A huge reservoir of this
zoonotic pathogen could be building up in the cattle population from infected
calves through consumption of contaminated water. The farm from which these
isolates were obtained is stocked with imported exotic breeds from countries
where E. coli 0157:NM has been incriminated as a cause of epidemics (Armstrong
et al., 1996). Cattle, especially dairy cattle have been implicated as
the source of the organisms in outbreak situations (Allison et al., 2000;
Armstrong et al., 1996; Effler et al., 2001). Contamination of
water and food by organisms from bovine reservoirs is the major mode of transmission
to animals and man (Armstrong et al., 1996). Though the low percentage
of isolation is deceptive but this could have been due to the organisms
inability to survive for extended periods outside its host. Moreover, the infective
dose of STEC is extremely low. Consumption of food with 2 cells/25 g has been
reported to have caused illness in man (Sekla et al., 1990). Studies
have shown that cattle can habour the organism without showing any overt or
clinical signs including faecal shedding (Armstrong et al., 1996; Effler
et al., 2001). Shedding has been shown to commence when the host is subjected
to stress such as changes in diet and drought (Effler et al., 2001).
Effler et al. (2001) reported that E. coli 0157:NM could not be
found in South African waters one year after the outbreak due to its inability
survive outside its natural host.
The finding that the two isolates were resistant to ampicillin, streptomycin, sulphamethoxazole and tetracycline has serious implication on the continued use of sub-therapeutic levels of these drugs in animal production, particularly where these un-recommended levels are often used by feed manufacturers as growth promoters. This use has often been blamed for the wide spread resistance of bacteria to antimicrobial agents in animals and man (Weber and Courvalin, 2005). The result of this study, though very limited supports this view since 100% resistance was observed to antimicrobials ampicillin, streptomycin, tetracycline, compound sulphonamides and sulphamethoxazole used commonly in animal production and veterinary practice in Zaria (Ramon-Yusuf et al., 1990). Kabir et al. (2002) reported that the failure to observe withdrawal time, un-recommended drug usage and disregard of meat inspection procedures and laws, are responsible for the abuse/misuse of drugs in ruminant and intensive poultry production in Zaria. This may have contributed greatly to the resistance exhibited by the organism to the drugs.
Resistance to nalidixic acid is inherent, caused mostly by mutations and is important mainly from a public health view point, because it is the type quinolone- a class of frontline antibiotics that offer hope in infectious disease therapy against resistant pathogens. This may explain the resistance since nalidixic acid is not recommended for therapeutic purposes in veterinary practice. The quinolones are antibacterial agents that target two essential bacteria enzymes; DNA gyrase and DNA topoisomerase IV. These enzymes are essential in DNA synthesis in bacterial and by preventing their production quinolones destroy bacterial cell walls leading to death of the bacteria. Mutations remove the site of quinolone activity from the gryA and gryB genes of E.coli. These mutations can be induced by substances and proteins that posses quinolone- like activity such as bacteria proteins like Microcin B17 (Drlica and Zhao, 1997), making the organisms resistant to quinolones.
The two isolates predictably showed high sensitivity to the flouroquinolones used. The fluorinated quinolones such as norfloxacin and ciprofloxacin are exceptionally potent in attacking bacteria, because their action is at several sites and thus avoiding point specific mutations effect associated with quinolone action (Ozeki, 1997). Sensitivity to chloramphenicol may be due to the fact that it is not recommended for use in food animals and is unavailable in preparation suitable for veterinary use in northwestern Nigeria. This viewpoint is supported by Kabir et al. (2000) and Ramon-Yusuf et al. (1990) who have reported that resistance to antimicrobial agents is strongly linked to abuse/misuse in animal health and production practices. Sensitivity to fluoroquinolones and cloramphenicol is an important finding from a public health viewpoint. These drugs are available in Nigeria and are fairly affordable since they are manufactured locally. However, it is important to chlorinate water that is used for human consumption and to prevent faecal contamination of water that is intended for cattle consumption to forestall the outbreak in the human population and the devastating effect that outbreaks can have on the cattle industry. Effler et al. (2001) reported that the South African and Swaziland outbreak wiped out calves and young stock on most farms in the affected areas.
To control the spread of resistance we must heed the warning of Alexander Fleming in his 1945 Noble Prize acceptance speech: Dr. Fleming noted the danger of sub-therapeutic use of antimicrobial agents and said. If you use penicillin, use enough (Fleming, 1945).
This study has exposed the magnitude of the problem of indiscriminate importation of livestock and the association between importation and emergence of new pathogens. Escherichia coli 0157: NM is present in water sources consumed by cattle in northwestern Nigeria. The future of food safety and security lies in education and legislation. Education is required so that the public is aware of the risk associated to certain animal production practices like indiscriminate drug use and its consequences. Legislation is required to deter offenders concerning the sale, acquisition and administration of antimicrobial agents in animals.
We express our gratitude to Dr. Stella I. Smith, National Institute of Medical Research Yaba, Lagos for providing the control strain-ATTC 43895. Our appreciation is also extended to the National Veterinary Research Institute (NVRI) Vom, Plateau State, for the technical supports we received for the assays. This study was supported in part by a grant from the Board of Research, Ahmadu Bello University, Zaria, Nigeria.
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