Antimicrobial-resistant Klebsiella species isolated fromfree-range chicken samples in an informal settlement
Burtram C. Fielding1, Amanda Mnabisa2, Pieter A. Gouws3, Thureyah Morris2
1Molecular Virology Laboratory, Medical Microbiology Cluster, Department of Medical
Biosciences, Faculty of Natural Sciences, University of the Western Cape, South Africa
2Food Toxicology Laboratory, Medical Microbiology Cluster, Department of Medical
Biosciences, Faculty of Natural Sciences, University of the Western Cape, South Africa
3Food Microbiology Laboratory, Department of Biotechnology, Faculty of Natural
Sciences, University of the Western Cape, South Africa
Bellville, Western CapeSouth Africa 7535Phone: +27-21-9592331
Fax: +27-21-9593125E-mail: firstname.lastname@example.org
Introduction: Sub-therapeutic doses of antimicrobial agents are administered
routinely to poultry to aid growth and to prevent disease, with prolonged expo-sure often resulting in bacterial resistance. Crossover of antibiotic resistant bac-teria from poultry to humans poses a risk to human health. Material and methods: In this study, 17 chicken samples collected from a ven-dor operating in an informal settlement in the Cape Town Metropolitan area,South Africa were screened for antimicrobial-resistant Gram-negative bacilliusing the Kirby Bauer disk diffusion assay. Results: In total, six antibiotics were screened: ampicillin, ciprofloxacin, gen-tamicin, nalidixic acid, tetracycline and trimethoprim. Surprisingly, Klebsiellaozaenae was identified in 96 and K. rhinoscleromatis in 6 (n = 102) of the sam-ples tested. Interestingly, ~40% of the isolated Klebsiella spp. showed multipleresistance to at least three of the six antibiotics tested. Conclusions: Klebsiella ozaenae and K. rhinoscleromatis cause clinical chronicrhinitis and are almost exclusively associated with people living in areas of poorhygiene.
Key words: antibiotic-resistant, poultry, resistance, Klebsiella species.
Antimicrobial agents are frequently used in food animals for the pro-
motion of growth . The latter is achieved by the antibiotics decreasingthe susceptibility of the animals to bacterial infections and by increasingfood absorption in the intestine. Of great concern is the fact that theseantimicrobial agents are the same, or closely related, to antimicrobialsused in human medicine . Also, as a result of the widespread use ofthese antimicrobial agents, bacteria have developed ways to circumventthe effects of antibiotics through evolutionary adaptations .
Therefore, contamination of food with antibiotic-resistant bacteria pos-
es a major threat to public health and the transfer of these antibiotic-resis-tant traits to pathogenic bacteria could potentially compromise the treat-ment of bacterial infections in the clinical setting .
Members of the genus Klebsiella, especially K. pneumonia and K. oxy-toca, are opportunistic pathogens associated with severe nosocomial infec-
Burtram C. Fielding, Amanda Mnabisa, Pieter A. Gouws, Thureyah Morris
tions such as septicaemia, pneumonia and urinary
Identification of Klebsiella spp.
tract infections. K. pneumonia has been taxonom-
After the overnight incubation, one isolate from
ically subdivided into three subspecies: K. pneu-
each of the three XLD and BGA plates was select-
monia subsp. pneumonia, K. pneumonia subsp. oza-
ed and emulsified in 5 ml of sterile saline solution. enae and K. pneumonia subsp. rhinoscleromatis .
The isolates were identified using the MicrobactTM
Klebsiella rhinoscleromatis and K. ozaenae cause
12B identification kit according to the manufactur-
two clinical forms of chronic rhinitis, namely
rhinoscleroma and ozena, respectively . Both dis-eases are endemic in areas with poor hygiene con-
ditions and are commonly not detected in devel-oped countries [6, 7]. Even though the isolation of
Isolates were screened for antibiotic resistance
antimicrobial-resistant and -susceptible strains of
using the Kirby-Bauer disk diffusion assay. For each
Klebsiella from poultry have previously been report-
sample, three colonies were selected from both XLD
ed [8, 9], this is, to our knowledge, the first report
and BGA plates respectively, and inoculated into
of the isolation of multiple-antibiotic-resistant Kleb-
Tryptone Soya Broth (TSB) (Oxoid, CM129). Thus,
siella rhinoscleromatis and K. ozaenae isolated from
six isolates were obtained from each sample to
ensure reproducibility of the results. The cultureswere incubated for 4 h at 37°C with continuous
shaking. A 100 μl of inoculum was then spread onto
Mueller-Hinton Agar (MHA) (Oxoid, CM0337). Antibi-otic disks were dispensed onto the plates and incu-
Free-range chicken samples (n = 17) were col-
bated overnight at 37°C. Isolates were tested for
lected from street vendors from the informal set-
susceptibility to six different antimicrobial agents,
tlement of Langa, in the Western Cape Province of
namely ampicillin (10 μg), ciprofloxacin (5 μg), gen-
South Africa. Neck-skin samples were collected
tamicin (10 μg), nalidixic acid (30 μg), tetracycline
aseptically in sterile sample bags, refrigerated upon
(30 μg) and trimethoprim (1.25 μg). The CLSI 
arrival in the laboratory and processed for micro-
guidelines were utilized to classify the isolates as
(i) resistant, (ii) intermediate-resistant or (iii) sus-ceptible to the various antibiotics. Isolates exhibiting
intermediate resistance were classified as resistant.
Twenty-five grams of neck skin was aseptically
weighed and placed into a sterile polyethylene
stomacher bag containing 225 ml of Buffered Pep-
The antibiotic code profile developed by Manie
tone Water (BPW) (Oxoid, CM0509). Each sample
et al.  was used to determine whether the iso-
was homogenized for 120 s at normal speed using
lates were displaying multiple antibiotic resistance
a Seward stomacher. One ml of the homogenate
(MAR) (see supplemental data for explanation).
was then transferred into 9 ml sterile BPW and 10-fold serial dilutions were performed (10–1-10–6).
A 100 μl aliquot of each dilution was then spread-plated onto plate count agar (PCA) and incubated
In this study, of the 102 Enterobacteriaceae iso-
for 18 h at 37°C. Following overnight incubation,
lated using conventional selective media, 96 were
bacterial colonies were enumerated to determine
identified as Klebsiella ozaenae and 6 as K. rhino -scleromatis with the Microbact 12B identification
Additionally, after 6 h of incubation at 37°C, kit. The resistance of these Klebsiella spp. to six
100 μl of the pre-enriched sample was pipetted
antibiotics was determined using the Kirby-Bauer
from each dilution series into 9.9 ml Rappaport-
disk diffusion assay. Isolates showed high levels of
Vassiliadis soya peptone broth (RV) (Oxoid,
resistance; 66.7% were resistant to ampicillin, 61.8%
CM0669) and incubated overnight at 37°C. After
to nalidixic acid, 59.8% to tetracycline and 50% to
24 h of incubation, growth in tubes with the high-
trimethoprim (Table I). In fact, of greater concern,
est dilution were selected from each sample and
the antibiotic code profiles showed that ~40% of
streaked onto three Xylose Lysine Deoxycholate
the isolated strains showed multiple antibiotic
(XLD) (Oxoid, CM0469) plates and three Brilliant
resistance (MAR) to three or more antibiotics: (i)
Green Agar (BGA) (Oxoid, CM0263) plates. These
21.5% of the Klebsiella spp. showed resistance to
respective selective media allow for the presump-
ampicillin, trimethoprim, nalidixic acid and tetracy-
tive identification of Enterobacteriaceae; XLD also
cline, (ii) 9.8% showed resistance to ampicillin,
differentiates between pathogenic Enterobacteri-
nalidixic acid and tetracycline and (iii) 8.8% showed
aceae. The agar plates were then incubated
resistance to ampicillin, trimethoprim and nalidixic
Antimicrobial-resistant Klebsiella species isolated from free-range chicken samples in an informal settlement
Table I. Antimicrobial resistance patterns of Klebsiella
otics that are active on Gram-negative bacteria
except for penicillin, ampicillin and sulfonamide, with ciprofloxacin and rifampin the most effec-
tive antimicrobials ; in terms of efficacy/cost,
the association of trimethoprim-sulfamethoxazole
is the best compromise . Botelho-Nevers et al. also reported good results when using rifampin
associated with co-trimoxazole to treat K. rhinoscle-romatis. Limited antimicrobial susceptibility stud-
ies of K. ozaenae have shown varying degrees ofsusceptibility to ampicillin [13, 16, 17].
There is evidence that the routine use of antibi-
Table II. The multiple antibiotic resistance code profile
otics in animal husbandry leads to antibiotic resist-
of Klebsiella spp. isolated from free-range chickens
ance in bacteria. These antibiotic-resistant bacteriacan infect or reach the human population not only
by direct contact, but also by food products of ani-
mal origin . Therefore, the reduction and even-
tual elimination of antibiotics for purposes other
than veterinary therapy or treatment of infectionsin animals is essential. This can be achieved by
improving methods of animal husbandry, the erad-
ication of diseases in animals, the optimal use of
existing vaccines and the development of new vac-cines [18, 19]. Generally, these interventions are
aimed at reducing the development and incidence
of resistant bacterial infections, thereby prolonging
or restoring the effectiveness of existing antibiotics. Importantly, a good programme for prevention
of antibiotic resistance also includes an active sys-
tem of surveillance for resistance, an active and
effective infection control programme to minimizesecondary spread of resistance, and the sensible use
of antimicrobials in animal production systems .
This manuscript reported the isolation of multi-
ple-antibiotic-resistant K. rhinoscleromatis and
K. ozaenae from chicken samples collected froma vendor in an informal settlement in South Africa.
Surprisingly, resistance to antibiotics commonly
used in the clinical treatment of K. rhinoscleroma-tis and K. ozaenae infection was observed. Thiscould pose a serious health risk if vertical trans-
mission occurs between infected poultry samplesand humans.
Antibiotics, such as bacitracin, chlortetracycline,
erythromycin and penicillin, are routinely used forcontrol and treatment of bacterial diseases in poul-
The authors received funding from the Univer-
try. When these antibiotics are administered to the
sity of the Western Cape Senate Research Fund.
birds over extended periods, especially at low lev-
Burtram C. Fielding also receives funding from the
els, certain species of bacteria become resistant .
National Research Foundation, South Africa.
Alarmingly, more and more scientific evidenceshows that these resistant bacteria, including
pathogens, can be transferred to humans throughthe food chain .
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