




































In ternationa l
Scholars
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African Journal of Pig Farming ISSN 2375-0731 Vol. 4 (11), pp. 001-009, November, 2016. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Characterisation of enterococci and Escherichia coli 

isolated from commercial and communal pigs from 

Mafikeng in the North-West Province, South Africa 
 

Moneoang, Modisane Simon1* and Bezuidenhout, Cornelius Carlos2
 

 
1
Department of Animal Health, North-West University (Mafikeng Campus), Private Bag X2046, Mmabatho, 2735, South 

Africa. 
2
School of Environmental Science and Development, North-West University (Potchefstroom Campus), Private Bag  

X6001, Potchefstroom, 2520, South Africa. 
 

Accepted 13 May, 2016 
 
The aim of this study was to isolate enterococci and Escherichia coli from faeces collected from commercial and 
communal pigs, and to characterise these isolates using antibiotic susceptibility profiles. Enterococcus selective agar 
and eosin methylene blue lactose agar were used for enterococci and E. coli isolation, respectively. Gram staining, 
API 20 Strep and API 20E were used for identification of enterococci and E. coli, respectively. Three-hundred-and-four 
enterococci and 208 E. coli were identified. The most prevalent enterococci species were Enterococcus faecium (58%) 
and Enterococcus gallinarum (23%). A large proportion of enterococci (62.5% to 100%) and E. coli (88.5 to 100%) were 
resistant to erythromycin, oxytetracycline and sulphamethoxazole. No vancomycin-resistant enterococci were found 
and PCR analysis for vanA, vanB and vanC-1 were all negative. Less than 7% of enterococci were resistant to 
ampicillin and amoxicillin, whereas 45% of E. coli isolates were resistant to the same antibiotics. Antibiotic 
susceptibility tests and clustering patterns showed some similarities among these isolates. From the results, a 
common origin of the isolates or histories of antibiotic use among these farms was proposed. It could also be 
concluded that vancomycin-resistant enterococci were not present in pigs on these two farms. 
 
Key words: Vancomycin resistant enterococci, vancomycin susceptible enterococci, tetracycline, resistance genes, 

multiple antibiotic resistance. 

 
INTRODUCTION 

 
Enterococci form part of the normal flora of the intestinal 
tract of animals and humans (Blondeau and Vaughan, 
2000; Klein, 2003). Among all the enterococci, Entero-
coccus faecium and E. faecalis are the most prevalent 
species (Takeuchi et al., 2005), and significant cause of 
nosocomial infections (Blondeau and Vaughan, 2000). 
Escherichia coli also form part of the normal microflora of 

the gut of humans and animals (Naylor et al., 2005). Pat-
hogenic E. coli strains have been recognised since the 
early 1900s, causing disease in both humans (Naylor et 
and animals (Blondeau and Vaughan, 2000).Multiple  
 
 

 
*Corresponding  author.  E-mail:  msmoneoang@nwpg.gov.za. 
Tel.: +2718 299 6800.Fax: +2718 293 3925. 

 
 
 
 
antibiotic resistance (MAR) is common among various 
microorganisms (Guan et al., 2002), and is nor-mally 
associated with the presence of antibiotic resistan-ce 
genes (Maynard et al., 2003; Bryan et al., 2004). Anti-
biotic susceptibility tests are used to generate MAR patt-
erns (Rota et al., 1996) and MAR indices (Kaspar et al., 
1990; Guan et al., 2002). These tests reflect the extent of 
selective pressures on the microbial floras of the gastro-
intestinal tract of humans and animals imposed by exces-
sive antibiotic use (Guan et al., 2002) . This technique 
could thus be used for classification and discrimination of 
isolates of the same or different location (Kaspar et al., 
1990; Guan et al., 2002).  
Enterococci have the ability to acquire and transfer re-

sistance genetic markers to and from other bacterial 
species such as Staphylococcus aureus (Gambaratto et - 



 
 
 

 

al., 2000). In the recent past, vancomycin has been used 
as the last resort to treat enterococcal infections (Gamba-
ratto et al., 2000). Vancomycin- resistant enterococci 
(VRE) are now endemic worldwide. It was firstly reported 
in Europe in 1986 and two years later in the United State 
of America (Perl, 1999). Furthermore, problems of VRE 
have also become a reality in South Africa (Derby et al., 
1998; von Gottberg et al., 2000).  

According to Blondeau and Vaughan (2000), resistance 
to vancomycin may occur due to mutations, transposons 
and resistance genes. Moreover, the extensive use of the 
growth promoter avoparcin in most of the European 
countries was also correlated to the high incidence of 
VRE (Wegener et al., 1999). In the United States, VREs 
were thought to occur primarily by the unskilful appli-
cation of vancomycin in hospitals (Lemcke and Bulte, 
2000).  

In most cases, resistance to vancomycin is associated 
with the activity of the vanA gene, which confers high 
levels of vancomycin and teicoplanin resistance (Fluit et 
al., 2001). On the other hand, vanB confers moderate 
levels of vancomycin resistance and susceptibility to tei-
coplanin in vitro (Fluit et al., 2001). Another vancomycin 
resistance determinant is VanC. The genes encoding for 
this are intrinsic in Enterococcus gallinarum (vanC- 1), E. 
casseliflavus (vanC- 2) and E. flavescens (vanC -3) (Fluit 
et al., 2001). In the clinical environment, it may be imp-
erative to determine the presence of these genes in order 
to establish the appropriate therapeutic and control mea-
sures (Hanaki et al., 2004). Studies have shown that 
PCR-based techniques are efficient and reliable for the 
surveillance of VREs (Bell et al., 1998; Fluit et al., 2001). 
The goal of this study was to isolate and characterise ent-
erococci and E. coli from a commercial and a communal 
pig farm in the North-West Province (RSA). The purpose 
was to determine the antibiotic resistance patterns of 
these isolates, and to screen for the presence of VRE or 
genes responsible for vancomycin resistance among the 
enterococci isolates. 
 

 
MATERIALS AND METHODS 

 
Sampling and methodology 

 
Two hundred faecal samples were collected from Mareetsane com-

mercial pigs and the same number collected from Tlapeng commu-
nal pigs. All the samples were collected using sterile gloves directly 

from the rectum. Faeces (1 g) was inoculated into nutrient broth (5 

ml) and incubated at 37°C for 24 h. These samples were sub-cul-

tured on Enterococcus selective agar and eosin methylene blue 

lactose agar (Merck, RSA) for enterococci and E. coli isolation, 

respectively. The plates were incubated at 37°C for 16 - 24 h. Colo- 
nies showing typical characteristics of entero-cocci (black) and E. 

coli (metallic sheen) were selected for further analysis. Gram Stain-

ing was performed, then API 20 Strep and API 20E (bioMêrieux, 

France) to identify enterococi and E. coli, respect-tively. 

 
 
 
 

 
Characterisation of isolates by MAR phenotypes and MAR 

indices 
 
The antibiotic susceptibility test was conducted by the disc -
diffusion method in Mueller-Hinton agar with and without 5% sheep 
blood for enterococci and E. coli , respectively (Kirby-Bauer, 1966) . 
The anti-biotics used are listed in Table 2 and abbreviations of 
antibiotics were according to the manufacturer (Mast Diagnostics, 
U.K.). Multi-ple antibiotic resistance phenotypes were determined 
for all the iso-lates that were resistant to three and more antibiotics 
(Rota et al., 1996). The MAR indices of isolates of a particular 
sampling station were also generated (Kaspar et al., 1990). 

 
DNA extraction and PCR 
 
Genomic DNA was extracted from selected MAR enterococci, E. 
coli and vancomycin-susceptible enterococci using a modified 
CTAB-PVP DNA extraction method (Doyle and Doyle, 1990). Brie-
fly, this included incubation in CTAB-PVP extraction buffer, followed 
by chloroform:isoamyl alcohol (24:1) extraction and precipitation of 
DNA using an ethanol and sodium chloride step. The extracted 
DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA [pH 
8.0]).  

The 16S rRNA primer pair (Table 1) was used to amplify 16S 
rRNA gene fragment (Positive control). The same DNA was then 
used for screening of vancomycin resistance gene fragments using 
primer pairs as indicated in Table 1. Primers were synthesised by 
Inqaba Biotech (Pretoria, RSA). All the amplifications were perfor-

med in a PTC-200 DNA Engine 
TM

 System (Bio-Rad, UK) . The 
PCR mix (25 µl) for amplification of 16S rRNA consisted of 100 ng 
of DNA containing 2X PCR master mix, 25 mM MgCl2, 16S primer 
(50 pmole), RNAse- DNAse free water (Fermentas, US) with the 
add-ition of 1 U Taq polymerase (Promega, USA). The PCR 
conditions consisted of an initial denaturing period of 5 min at 95°C, 
followed by 35 cycles of the following: 95°C for 30 s (denaturing), 
55°C for 30 s (annealing) and 72°C for 60 s (elongation), with a final 
elonga-tion step of 72°C for 5 min. 

For vancomycin resistance gene amplification, each PCR cycle 
consisted of an initial denaturing period of 2 min at 94°C, followed 
by 35 cycles of the following: 94°C for 60 s (denaturing), 62°C for 
60 s (annealing) and 72°C for 60 s (elongation), with a final elonga-
tion step of 72°C for 10 min (Bell et al., 1998). Another set of PCR 
reaction conditions were also tested and consisted of 35 cycles at 
94°C for 60 s (denaturing), 50°C for 60 s (annealing), 72°C for 60 s 
(elongation) and a final elongation step at 72°C for 10 min (Donabe-
dian et al., 2000).  

PCR products were resolved by gel electrophoresis on 1% (w/v) 
agarose that contained ethidium bromide (0.001 µg/µl). The electro-
phoresis buffer was 1X TAE (40 mM Tris, 20 mM acetic acid, 1 mM 
EDTA). Electrophoresis was performed at 60 V for 60 min. A Gene 
Genius Bio- Imaging System (Syngene, Synoptics, UK) was used to 

capture the image using GeneSnap (version 6.00.22) software. 

 
Statistical analysis 
 
The complete antibiotic inhibition zone data set of both enterococci 
and E. coli were separately analysed using Minitab Release (ver-
sion 13.31, France). These data sets were subjected to Pearson’s 
correlation to determine the relationship of all the enterococci and 
E. coli isolated from the two farms. The correlation was considered 
significant at P<0.01. Furthermore, a pooled data set of the total anti-
biotic inhibition zone diameters for E. faecium and E. coli were exported 
to Statistica (version 7; StatSoft software, US). The data were analysed 
by multivariate exploratory techniques using the Ward’s clustering 
method and Euclidean distances. The resultant dendrogram indicated 

clustering patterns and relationships (Berge et al., 2003). 



 
 
 

 
Table 1. Oligonucleotide primers and their sequences used in this study.  

 
 Primer Gene fragment  Sequence  Reference 

 

 VanAF   5 GTA GGC TGC GAT ATT CAA AGC 3   
 

 VanAR vanA  5 CGA TTC AAT TGC GTA GTC CAA 3  
Bell et al., 1998 

 

 

VanBF 
  

5 GTA GGC TGC GAT ATT CAA AGC 3 
 

 

     
 

 VanBR vanB  5 GCC GAC AAT CAA ATC ATC CTC 3   
 

 VanC1F   5 TGG TAT TGG TAT CAA GGA AAC C 3   
 

 VanC1R vanC-1  5 AGA TTG GAG CGC TGT TTT GTC 3   
 

 GM5F   5 TAC GGG AGG CAG CAG 3  
Muyzer et al., 1995 

 

 907 R 16S  5 CCG TCA ATT CCT TTG AGT TT 3  
 

    
 

 

 
Table 2. The numbers and percentages of various enterococci species and E. coli isolated from Mareetsane commercial 

pigs and Tlapeng communal pigs that were resistant to different antibiotics.  
 

Mareetsane commercial pigs   
 Microorganis    AP  A  E OT  SMX KF NE GM  VA C 

 

 m    10µg  10µg  15µg 30µg  200U 30µg 30µg 30µg  30µg 30µg 
 

 E. faecium  NR 3 1 64 86 86 74 58 75 0 - 
 

 NT = 86 % 3.5 1.2 74.4 100 100 86.0 67.4 87.2 0 - 
 

 E. gallinarum  NR 2 1 24 30 30 21 19 27 0 - 
 

 NT = 31 % 6.5 3.2 77.4 96.8 96.8 67.7 61.3 87.1 0 - 
 

 E. durans  NR 0 0 2 8 8 0 2 5 0 - 
 

 NT = 8 % 0 0 25 100 100 0 25 62.5 0 - 
 

 E. avium  NR 0 0 4 6 6 1 2 4 0 - 
 

 NT = 6 % 0 0 66.7 100 100 16.7 33.3 66.7 0 - 
 

 E. coli  NR 29 31 95 99 92 23 33 8 - 18 
 

 NT = 104 % 27.9 29.8 91.3 95.2 88.5 22.1 31.7 7.7 - 17.3  

  
 

          Tlapeng communal pigs     
 

 E. faecium  NR 1 0 45 57 64 43 46 44 0 - 
 

 NT = 64 % 1.6 0 70.3 89.1 100 67.2 71.9 68.8 0 -  

  
 

 E. coli  NR 8 8 46 46 46 4 0 0 - 2 
 

 NT = 46  %  17.4  17.4  100 100  100 8.7 0 0  - 3 
 

 
NT (number tested), NR (number resistant), A (amoxicillin), AP (ampicillin), GM (gentamycin), OT (oxytetracycline), NE (neo-

mycin), SMX (sulphamethoxazole), KF (cephalothin), E (erythromycin), VA (vancomycin), C (chloramphenicol). 
 

 

RESULTS 

 

Bacterial species identification 

 

Three-hundred-and- four (76%) enterococci and 208 
(52%) E. coli were positively identified from 400 faecal 
samples. During this study, E. faecium was the dominant 
species isolated and constituted 48.5% (Tlapeng) and 
58% (Mareetsane) of the total enterococci population. 
The level of E. gallinarum was 9% among the Tlapeng 
isolates and 23% among the Mareetsane isolates. The 
other enterococci species contributed between 0.5 and 
4% to the total enterococci population from both pig 

 
 

 

farms and the rest could not be identified to species level. 
 

 

Antibiotic susceptibility tests 

 

The activities of nine different antibiotics were tested on 
both enterococci and E coli, and the results are represen-

ted in Table 2. A large proportion of enterococci isolates 
(62.5 - 100%) showed resistance to oxytetracycline, sul-
phamethoxazole, gentamycin and erythromycin. Among 
the enterococci, 70.3 to 100% of E. faecium isolated from 
both farms were resistant to erythromycin, oxytetracycline 
and sulphamethoxazole. A considerable percentage of 



 
 
 

 

these E. faecium isolates were resistant to aminogly-
cosides (67.4 - 87.2%) and cephalothin (67.2 - 86.0%). 
Less than 7% of enterococci isolates were resistant to - 
lactam antibiotics (ampicillin and amoxicillin). All the 
tested enterococci from Mareetsane and Tlapeng pigs 
were susceptible to vancomycin.  

To establish whether the genes responsible for vanco-
mycin resistance (particularly low levels) might be pre-
sent in the enterococci populations from Mareetsane and 
Tlapeng pig farms, PCR assays were employed. The 
genes targeted included vanA, vanB and vanC-1 . All the 
identified E. faecium were screened for presence of vanA 
and vanB genes, and 50% (8 from 16) of E. gallinarum 
from both pig farms were screened for presence of vanC-
1 gene. None of the enterococci species tested harb-
oured either the vanA, vanB and/or vanC -1 genes (res-
ults not shown). However, only an image of the positive 
control (16S rRNA amplicons) was depicted in Figure 3.  

Susceptibility profiles for E. coli showed that the majo-

rity (88.5 - 100%) was resistant to erythromycin, oxytetra-
cycline and sulphamethoxazole (Table 2). Similar results 
were observed in enterococci isolates (Table 2). Levels of 
-lactam (ampicillin, amoxycillin and cephalothin)-resis-tant 
E. coli isolated from faeces of pigs in Mareetsane and 

Tlapeng ranged from 17.4 to 29.8% (Table 2). 

 

MAR phenotypes and MAR indices 
 
During this study, 150 E. faecium (86 and 64) and 122 E. 
coli (76 and 46) isolated from Mareetsane commercial 
and Tlapeng communal pig farms, respectively, were 
characterised for the MAR phenotypes and MAR indices. 
Ten dominant MAR phenotypes were observed from the 
150 E. faecium tested. The predominant MAR phenotype 
was E-OT-SMX-KF-NE which occurred in 36% of the 
Mareetsane isolates, and E- OT-SMX-KF in 43.8% of the 
Tlapeng isolates. Thus, the majority of E. faecium iso-
lates from both farms were resistant to E-OT-SMX -KF. 
The MAR indices were almost similar for Mareetsane and 
Tlapeng E. faecium isolates (0.5503 and 0.5121, respec-
tively). Six dominant MAR phenotypes were found among 
122 E. coli isolated from Mareetsane and Tlapeng pigs. 
The predominant MAR phenotype was E-OT-SMX, occ-
urring in both Mareetsane (36.8%) and Tlapeng (65.2%) 
isolates. The MAR indices for Mareetsane and Tlapeng 
E. coli isolates were 0.4369 and 0.3864, respectively. 

 

Clustering analysis 
 
A total of 92 E. faecium isolates, as well as 92 E. coli 

were selected from both Mareetsane and Tlapeng pigs 
(46 per sampling station) in order to generate a dendro-
gram. Among all 46 isolates selected, 23 were from the 
dominant MAR phenotypes. The other 23 isolates were 
from the least dominant MAR phenotypes. This selection 
criterion was decided upon to maximise the potential for 

 
 
 
 

 

determining similarities and differences between the two 
pig farms. The results of the clustering analysis for both 
E. faecium (92) and E. coli (92) are presented in Figures 
1 - 3, respectively. Figure 1 indicates 5 major clusters 
from the 92 E. faecium isolated from Mareetsane and 
Tlapeng pigs. Cluster I and III were mostly represented 
by Mareetsane isolates (78.3 and 57.1%, respectively). 
Clusters IV and V were represented mostly by Tlapeng 
isolates (78.9 and 76.9%). However, cluster II was for-
med by equal proportions (50%) of E. faecium from both 
farms, suggesting a close relationship of these isolates. 
Four major clusters were observed from E. coli isolated 
from both farms (Figure 2). Briefly, cluster I was mainly 
made up of Mareetsane isolates (93.8%), cluster II and 
cluster IV of Tlapeng isolates (73.1 and 63.2%, respec-
tively). In cluster III a mixture of Mareetsane and Tlapeng 
isolates (54.8 and 45.2%), respectively, were found. 
 

 

DISCUSSION 
 
During this study enterococci and E. coli were isolated 
from faeces collected from both commercial and commu-
nal pigs. However, the numbers of these isolates were 
lower than the number of samples collected, since APIs 
could not confirm the identities of some of the other puta-
tive isolates. Results such as these are not uncommon 
and have also been reported previously (Hayes et al., 
2003).  

E. faecium was the most dominant enterococcal spe-
cies identified in this study. The observation of this study 
is in agreement with Klein (2003) in which E. faecium was 
the most frequent species isolated from pigs. High levels 
of E. faecium were also observed among the 981 retailed 
raw meat (chicken, turkey, pork and beef) samples ran-
domly obtained from 263 grocery stores in Iowa. Among 
all these meat samples, 61% of the isolates were E. 
faecium, 29% E. faecalis and 0.7% E. gallinarum (Hayes 
et al., 2003).  

A large proportion of enterococci were resistant to mul-
tiple antibiotics, with resistance mostly shown to erythro-
mycin, oxytetracycline and sulphamethoxazole. These 
results suggest a possibility of high use or exposure of 
these antibiotics within these pig farms. Less than 7% of 
all the enterococci isolates were resistant to -lactams 
(ampicillin and amoxicillin), indicating that these products 
could still be useful for enterococcal infection, particularly 
on the farms sampled.  

During this study, large proportions of enterococci were 
resistant to the macrolide antibiotic (erythromcin). How-
ever, all of these isolates were susceptible to glycopep-
tide (vancomycin). In most of the European countries, the 
prevalence of erythromycin- and vancomycin-resistant 
bacterial species is mostly associated with the use of 
growth promoters such as the macrolide, tylosin (Boerlin 
et al., 2001) and the glycopeptide, avoparcin (Wegener et 
al., 1999). Additionally, studies have shown that cross- 



 
 

 
Tree Diagram for 92 Cases   

Ward`s method  
Euclidean distances  

9MCC 
11MCCA  
51MCCA 

11TCN  
22MCCA 
2MCCA  
35TCN 
89MCC 

5MCC 
30MCC  

3MCC 
87MCC 
24MCC 
26TCN 
24TCN  
7MCC 

64TCN 
15TCN 
36MCC 

4TCN 
33TCN 

98MCCA 
48MCCA 
33MCCA 

33MCC 
13TCN 

8MCCA 
38MCC 

1TCN  
2TCN 

40TCN 
12TCN  

61MCCA 
47TCN  

32MCCA 
46TCN 
10TCN 
88MCC 
44MCC 
43MCC  

2MCC 
84MCC 
10MCC 
79MCC 

6MCCA 
15MCC 
42MCC 
45MCC 

8TCN 
6MCC 
7TCN 

59MCCA 
63MCCA 

41MCC  
83MCC 
53TCN 
19MCC 
18MCC 
16TCN 
54TCN 

84MCCA 
74MCCA 
69MCCA  

21TCN 
80MCCA 

9TCN 
25TCN 
48TCN 
38TCN 
34TCN 
19TCN 

41TCN 
6TCN 

3TCN 
20TCN 
45TCN 
39TCN 
36TCN 
28TCN  

47MCCA 
32TCN  

38MCCA 
56TCN  
60TCN 
27TCN  

34MCCA 
55TCN 
75TCN 
57TCN 
58TCN 
61TCN 
59TCN  

 
 
 
 
 

 
Cluster I 

 
 
 
 
 

 

Cluster II 
 
 
 
 
 
 
 
 
 
Cluster III 
 
 
 
 
 
 
 
 

 

Cluster IV 
 
 
 
 
 
 
 
Cluster V 

 

0 50 100 150 200 250 300 
 

(Dlink/Dmax)*100 
 

Figure 1. A dedrogram showing clustering patterns for 92 E. faecium isolated from Mareetsane and 

Tlapeng pigs. The analysis is based on complete antibiotic inhibition zone diameter data. Designation: 

MCCA; MCC (Mareetsane isolates), TCN (Tlapeng isolates). 
 

 

selection and genetic linkage between erythromycin and 
vancomycin resistance occurs particularly when tylosin is 

used as a growth promoter (Boerlin et al., 2001; Jackson 
et al., 2004). E. faecium from these pigs were resistant to 

 
 

 

both erythromycin and vancomycin (Boerlin et al., 2001). 

In our study, a large proportion of enterococci isolates 
from both farms were resistant to erythromycin. This is a 

cause for concern. Even so, all the tested enterococci 



                         
 

               Tree Diagram for 92 Cases       
 

                Ward`s method         
 

                Euclidean distances         
 

 

9MCCA 
                           

 

                            
 

 7MCCB                             

 76MCCB                            
 

 
66MCCB                            

 

 11MCC B               

Cluster I          

 18MCCB              

 

         

                      

 79MCCB                            
 

 36TCN                             

 77MCCB                            
 

 57MCC B                             

 88MCCA                            
 

                            

 6MCCB                            
 

 
15MCC B                            

 

 56MCCB                            
 

 
23MCCB                             

                            

 59MCCB                             

                            

 92MCCB                            
 

 61MCCB                            
 

 49MCCB                            
 

 
99TCN                             

                            

 82MCCB                            
 

 42TCN                             

 97TCN                            
 

                           
 

 93TCN                            
 

 
81TCN                            

 

 89TCN                             

 44TCN                

Cluster II 
        

 

                       
 

 27MCCB                         

 2MCCB                             

 19MCCB                            
 

 1TCN2                             

 91TCN                            
 

 GTCN1                            
 

 KTCN2                            
 

 FTCN                            
 

 22TCN                             

 56TCN                            
 

 
ATCN1                            

 

 24TCN                            
 

 
ATCN2                             

                            

 UTCN1                            
 

 XTCN1                             

 43MCCA                            
 

 1MCCA                             

 21MCCB                            
 

100MCCA                             

 29MCCB                            
 

 90TCN                             

 98TCN                            
 

                            

 32MCCB                            
 

 25MCCB                            
 

 22MCCB                            
 

 
1TCN                            

 

 
24MCC B                            

 

 MTCN                             

 47TCN                  Cluster III       
 

 28TCN                         

 YTCN1                            
 

 44TCN1                            
 

 LTCN2                             

 VTCN1                             

                           
 

 

93MCCB 
                            

                           
 

 11MCC A                             

 12MCCA                            
 

 CTCN                             

 ETCN2                            
 

                            

 27MCCA                            
 

 15MCCA                             

 17MCCB                            
 

 3MCCB                             

 

10MCCA 
                            

                           
 

 9TCN                             

 79TCN                            
 

 84MCCA                            
 

 
34MCCB                            

 

 2MCCA                            
 

 28MCCB                            
 

 14MCCB                            
 

 95TCN                

Cluster IV         
 

 94TCN                        
 

                        

 14MCCA                            
 

 21TCN                             

 
RTCN                             

                            

 12MCCB                            
 

 100TCN                            
 

 
25TCN                             

                            

 96TCN                            
 

 43TCN                             

 82TCN                            
 

 6TCN                             

 79TCN2                            
 

 TTCN1                            
 

0 20 40 60 80  100 120 140 160  
   

(Dlink/Dmax)*100 
 

Figure 2. A dendrogram showing clustering patterns for 92 E. coli isolated from Mareetsane and 

Tlapeng pigs. The analysis is based on complete antibiotic inhibition zone diameter data. 

Designation: MCCA; MCCB (Mareetsane isolates), TCN, TCN1, TCN2 (Tlapeng isolates). 
 

 

from Mareetsane and Tlapeng pigs were susceptible to 
vancomycin. The absence of VRE is not uncommon. 
Vancomycin - susceptible enterococci (VSE) were obser-
ved from faecal samples collected from pigs in Sweden 
(van den Bogaard et al., 2000). Such a trend was also 

 
 

 

observed among enterococci isolated from retailed raw 
meat products in Iowa (Hayes et al., 2003). 

The farmers from both Mareetsane commercial and 

Tlapeng communal settings indicated that they do not use 

any growth promoters (tylosin and/or avoparcin) on 



 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Image of a composite agarose gel showing an amplified 16S rRNA gene 

fragment from enterococci and E. coli. Lane 1 (Lambda DNA-Hind III digest), Lanes 2-3 

(16S rRNA; enterococci), Lanes 4-5 (16S rRNA; E. coli). 
 

 

their farms. They also indicated that they do not use 
vancomycin or any glycopeptides antibiotics for therapeu-
tic purposes. Thus, the results from this study support 
their claims. When antibiotic resistance data sets of E. 
faecium isolated from the two farms were compared, a 
strong positive correlation (r = 0.992; p < 0.001) was obs-
erved between these data sets. This result suggested 
that pigs from these two farms had a common history of 
antibiotic exposure.  

Commercial and communal farmers in the North-West 
Province, particularly those from areas around Mafikeng, 
have indicated that they rely on tetracycline for thera-
peutic treatment of their animals. Tetracycline is attractive 
to these farmers not only because of being a broad spec-
trum antibiotic, but also because it is a freely available 
over-the-counter product which is cost-effective. The res-
ults of the present study underscore the hypothesis that 
the continuous use of an antibiotic results in bacterial 
tolerance to that product. As such, high numbers of E. 
coli resistant to this antibiotic were isolated. This poses a 

potential threat to both animals and human health sec-
tors.  

In a study conducted in Canada, E. coli isolated from 
pigs were resistant to oxytetracycline (Maynard et al., 
2003; Bryan et al., 2004). Previous studies showed that a 
considerable large percentage of E. coli isolated from ani-
mals such as chickens, cattle, goats and sheep (Bryan et 
al., 2004), as well as in human isolates (Bartoloni et al., 
2006) were resistant to tetracycline.  

A considerable percentage of E. coli isolated from pigs 

from both farms showed resistance to ß-lactam antibio- 

 
 

 

tics (Table 2). Maynard et al. (2003) observed that bet-
ween 15 - 30% of ETEC strains isolated from pigs in 
Canada were also resistant to these ß-lactams. Resist-
ance to such antibiotics among E. coli isolated from pigs 
from Mareetsane and Tlapeng could be due to mutations 
in the penicillin-binding proteins and/or presence of 

blaTEM resistance genes (Fluit et al., 2000). The higher 

percen-tages (29.8%) of these E. coli were resistant to 
the newer generation ß-lactam antibiotic, cephalothin, 
and this is a cause for concern. When comparing 
resistance results of E. coli isolated from Mareetsane and 
Tlapeng pig farms, a strong positive correlation (r = 
0.992; p < 0.001) exis-ted, once again indicating common 
antibiotic usage regi-mes on these farms.  

During this study, a large percentage of enterococci 
and E. coli showed multiple antibiotic resistances. The 
most dominant MAR phenotype pattern was E-OT-SMX-
KF-NE for Mareetsane E. faecium isolates with only the 
absence of one antibiotic (NE) for Tlapeng isolates. The 
prevalent MAR phenotype for E. coli isolated from both 
pig farms was E-OT- SMX. During this study, it appeared 
that E-OT-SMX was the basis of all the phenotypes in 
both E. faecium and E. coli, suggesting a possibility of 
high use or exposure of these antibiotics in these pig 
farms. According to Kaspar et al. (1990), isolates with si-
milar MAR phenotypes may have common origin or simi-
lar antibiotic exposure histories. Furthermore, the MAR 
phenotypes may sometimes suggest the extent of anti-
microbials use in specific food animal production environ-
ments (Hayes et al., 2003).  

When comparing the relationship of both E. faecium 



 
 
 

 

and E. coli using the complete antibiotic inhibition zone 
diameter data by the clustering patterns (Figures 1 and 
2), some close relationship between these isolates from 
Mareetsane and Tlapeng existed. A similar situation was 
shown by these isolates when considering the results of 
the resistance patterns (Table 2), MAR phenotypes and 
MAR indices. These results support the deduction that 
the isolates from both farms may have a common origin. 
Alternatively, these isolates may have had similar histo-
ries of antibiotic exposures. 

 

Conclusion 
 
Enterococci and E. coli were isolated and characterised 
in this study. It could be concluded that VREs are 
currently not present in pigs on the farms that were 
tested. This observation is encouraging, particularly for 
the marketing of pork from these farms. However, further 
studies are essential to screen more farms in the North-
West Province to confirm the absence of VRE among 
pigs in this region. Furthermore, a large proportion of 
enterococci were susceptible to amoxycillin and ampi-
cillin. These results indicate that -lactams could still be 
used as a synergy with other cell wall- active antibiotics 
such as vancomycin in the treatment of enterococcal 
infections in pigs, particularly in the areas sampled in the 
North-West Province (RSA).  

The observations of this study provide some baseline 
data for management of antibiotic resistance among pigs 
in North-West Province (RSA). A large proportion of 
enterococci and E. coli showed MAR, and this should be 

considered a cause for concern for veterinary health aut-
horities. This also suggests the need for changes in the 
use of antibiotics in both veterinary and human medi-
cines, as some unusual events may affect propagation of 
antibiotic-resistant bacteria in the food environment and 
food supply. 

 

ACKNOWLEDGEMENTS 
 
This work was made possible by a grant from the Nation-
al Research Foundation (NRF) and funds from the Dep-
artment of Animal Health in the North-West University 
(Mafikeng Campus) . We also like to thank the farmers 
(Mr. Dryer and Mr. Molosi) for providing samples. Our 
acknowledgements are also extended to the staff mem-
bers, technicians, and colleagues within the Department 
of Animal Health, North-West University (Mafikeng Cam-
pus). 

 
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