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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: 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 [1]. 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 [1]. Also, as a result of the widespread use ofthese antimicrobial agents, bacteria have developed ways to circumventthe effects of antibiotics through evolutionary adaptations [2]. 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 [3]. 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 [4].
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 [5]. 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 [10] 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. [11] 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[14], with ciprofloxacin and rifampin the most effec- tive antimicrobials [15]; in terms of efficacy/cost, the association of trimethoprim-sulfamethoxazole is the best compromise [7]. 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 [18]. 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[20]. 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 [21]. 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 [9].
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 [12]. 1. Schroeder CM, Zhao C, DebRoy C, et al. Antimicrobial In the clinical setting, an adequate and pro- resistance of Escherichia coli O157 isolated from humans,cattle, swine, and food. Appl Environ Microbiol 2002; 68: longed antimicrobial regimen is needed to treat the two types of chronic rhinitis caused by K. ozaenae 2. Wright JG, Tengelsen LA, Smith KE, et al. Multidrug- and K. rhinoscleromatis [13]. Klebsiella rhinosclero- resistant Salmonella typhimurium in four animal facilities.
matis is normally sensitive to the majority of antibi- Burtram C. Fielding, Amanda Mnabisa, Pieter A. Gouws, Thureyah Morris 3. Van TT, Moutafis G, Istivan T, Tran LT, Coloe PJ. Detection of Salmonella spp. in retail raw food samples fromVietnam and characterization of their antibiotic resistance.
Multiple antibiotic resistance. Briefly, the antibi- Appl Environ Microbiol 2007; 73: 6885-90.
otics were divided into two groups and each antibi- 4. Brisse S, Verhoef J. Phylogenetic diversity of Klebsiella otic was designated a particular number. Group 1, pneumoniae and Klebsiella oxytoca clinical isolates which contained ampicillin, was designated num- revealed by randomly amplified polymorphic DNA, gyrA ber 1, ciprofloxacin was designated number 2 and and parC genes sequencing and automated ribotyping.
trimethoprim was designated number 4; group 2 Int J Syst Evol Microbiol 2001; 51: 915-24.
5. Malowany MS, Chester B, Allerhand J. Isolation and contained gentamicin and was also designated microbiologic differentiation of Klebsiella rhinoscleromatis number 1, nalidixic acid was designated number 2 and Klebsiella ozaenae in cases of chronic rhinitis. and tetracycline was designated number 4. If an isolate was resistant to a particular antibiotic it was 6. Medina L, Benazzo M, Bertino G, et al. Clinical, genetic given the number designated to that particular and immunologic analysis of a family affected by ozena.
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7. Evrard I, Gruyer X, Desse P, et al. Spheno-ethmoidal otic it was given zero. The numbers awarded were rhinoscleroma. Report of a case and review of the added to yield the respective code. For example, an literature. Ann Otolaryngol Chir Cervicofac 1998; 115: isolate resistant to ampicillin, ciprofloxacin and gen- tamicin, but sensitive to the other antibiotics would 8. Mamber SW, Katz SE. Effects of antimicrobial agents fed receive the code (1 + 2 + 0) (1 + 0 + 0) to give a pro- to chickens on some gram-negative enteric bacilli. Appl 9. Kilonzo-Nthenge A, Nahashon SN, Chen F, Adefope N.
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