Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin

İsmail Hakkı Tekiner Haydar Özpınar About the authors

Abstract

Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek® MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla -genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM & blaCTX-M (52.7%), blaTEM & blaSHV (20%), blaTEM & blaCTX-M & blaSHV (12.7%), and blaSHV & blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes.

Keywords
Bla gene; Enterobacteriaceae; ESBL; Public health; Turkey

Introduction

Antibiotics are used as veterinary and human medicines for treatment, control and prevention of infectious diseases. However, their repeated off-label over use can have un-anticipated adverse effects including development of antibiotic resistance in the bacteria towards modern beta-lactam antibiotics.11 Wang H, McEntire JC, Zhang L, Li X, Doyle M. The transfer of antibiotic resistance from food to humans: facts, implications and future directions. Rev Sci Tech. 2012;31:249-260.

One of the antibiotic resistance mechanisms in the bacteria is the production of specific enzymes such as beta-lactamase to break down the four-atom beta-lactam ring of the antibiotics. When this ring is opened through hydrolysis by these enzymes, the antimicrobial properties of the antibiotics are completely lost.22 Thenmozhi S, Moorthy K, Sureshkumar BT, Suresh M. Antibiotic resistance mechanism of ESBL producing Enterobacteriaceae in clinical field: a review. Int J Pure Appl Biosci. 2014;2:207-226. The extended spectrum beta-lactamases (ESBL) are the rapidly evolving plasmid-mediated group of beta-lactamases.33 Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev. 2005;18:657-686. ESBL can hydrolyse penicillins, first, second and third-generation cephalosporins, and aztreonam (but not cephamycins or carbapenems). The activity of ESBL can be inhibited by beta-lactamase inhibitors such as clavulanic acid (CLA).44 Rupp ME, Fey PD. Extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae: considerations for diagnosis, prevention and drug treatment. Drugs. 2003;63:353-365. The antibiotic resistance leads to increased morbidity, mortality and the cost of treating infections, in particular, those caused by ESBL-producing bacteria.55 EFSA. Scientific opinion on the public health risks ofbacterial strains producing extended-spectrum β-lactamasesand/or AmpC β-lactamases in food and food-producinganimals. EFSA J. 2011;9:2322–2417. The most prevalent extended spectrum beta-lactamases are TEM, SHV, and CTX-M variants.66 Liebana E, Carattoli A, Coque TM, et al. Public health risks of enterobacterial isolates producing extended-spectrum β-lactamases or AmpC β-lactamases in food and food-producing animals: an EU perspective of epidemiology, analytical methods, risk factors, and control options. Clin Infect Dis. 2013;56:1030-1037. The CTX-M variants are also clustered in five major groups: 1, 2, 8, 9, and 25.77 Bonnet R. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004;48:1-14. After 2000s, CTX-M type enzymes have exhibited a rapid growth over a wide range geographic areas, and have become much more prevalent than TEM and SHV type enzymes.88 Fernandes R, Amador P, Oliveira C, Prudêncio C. Molecular characterization of ESBL-producing Enterobacteriaceae in Northern Portugal. Sci World J. 2014;2014:782897, http://dx.doi.org/10.1155/2014/782897.
http://dx.doi.org/10.1155/2014/782897...

The ESBLs are mainly encoded by plasmids and mobile genetic elements such as integrons, insertion sequences, transposons and plasmids. These genetic elements are easily transferable to other bacteria such as those belonging to Enterobacteriaceae.55 EFSA. Scientific opinion on the public health risks ofbacterial strains producing extended-spectrum β-lactamasesand/or AmpC β-lactamases in food and food-producinganimals. EFSA J. 2011;9:2322–2417.,99 Giedraitienė A, Vitkauskienė A, Naginienė R, Pavilonis A. Antibiotic resistance mechanisms of clinically important bacteria. Medicina (Kaunas). 2011;47:137-146. The widespread use of third generation cephalosporins and aztreonam has led to the emergence and dissemination of ESBL producing strains and their encoding genes, in particular, among Enterobacteriaceae associated with severe enteric illnesses.1010 Mesa RJ, Blanc V, Blanch AR, et al. Extended-spectrum β-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage). J Antimicrob Chemother. 2006;58:211-215.1313 von Salviati C, Laube H, Guerra B, Roesler U, Friese A. Emission of ESBL/AmpC-producing Escherichia coli from pig fattening farms to surrounding areas. Vet Microbiol. 2015;175:77-84.

The major genes responsible for ESBL production include TEM genes (blaTEM), SHV genes (blaSHV), and CTX-M genes (blaCTX-M).1414 Habeeb MA, Sarwar Y, Ali A, Salman M, Haque A. Rapid emergence of ESBL producers in E. coli causing urinary and wound infections in Pakistan. Pak J Med Sci. 2013;29:540-544. This continued evolution is a serious threat to the public health by limiting the ability to treat bacterial infections.1515 Sharma M, Pathak S, Srivastava P. Prevalence and antibiogram of extended spectrum β-lactamase (ESBL) producing Gram negative bacilli and further molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. J Clin Diagn Res. 2013;7:2173-2177.,1616 WHO. Integrated surveillance of antimicrobial resistance: guidance from a WHO Advisory Group; 2013 ISBN 978 92 4 150631 1, Switzerland. Most of the medicines under human consumption are antibiotics. Moreover, antibiotic use in veterinary medicine and for growth promotion and disease prevention in agriculture, aquaculture and horticulture is also a huge problem of antibiotic resistance, since most of the antibiotics manufactured (100,000–200,000 tonnes per year) is estimated to be massively consumed by these sectors, especially in the food-producing animals for last 60 years.1717 Laxminarayan R, Duse A, Wattal C, et al. Antibiotic resistance – the need for global solutions. Lancet Infect Dis. 2013;13:1057-1098.,1818 Capita R, Alonso-Calleja C. Antibiotic-resistant bacteria: a challenge for the food industry. Crit Rev Food Sci. 2013;53:11-48. Therefore, food-producing animals and foods of animal-origin are under suspicion for being transmission vectors for colonisation and infection of the humans with ESBL-producing Enterobacteriaceae.1111 Reuland EA, al Naiemi N, Raadsen SA, Savelkoul PHM, Kluytmans JAJW, Vandenbroucke-Grauls CMJE. Prevalence of ESBL-producing Enterobacteriaceae in raw vegetables. Eur J Clin Microbiol Infect Dis. 2014;33:1843-2184.,1616 WHO. Integrated surveillance of antimicrobial resistance: guidance from a WHO Advisory Group; 2013 ISBN 978 92 4 150631 1, Switzerland.

The potential contribution of foods to human health risks by dissemination of plasmid-born ESBLs should be epidemiologically assessed because local, regional, national and international information related to this emerging phenomenon is largely incomplete in various geographical locations, including Turkey.99 Giedraitienė A, Vitkauskienė A, Naginienė R, Pavilonis A. Antibiotic resistance mechanisms of clinically important bacteria. Medicina (Kaunas). 2011;47:137-146.,1919 Arslan S, Özdemir F. Extended spectrum beta-lactamases in Escherichia coli strains isolated from homemade white cheeses: prevalence and antibiotics susceptibility. World J Microbiol Biotechnol. 2008;24:2361-2364. The Turkish authorities accept that the use of antibiotics in the food animals to make them grow faster and/or to prevent disease cannot be controlled effectively due to the economic benefits of food-animals, as the sector largely ignores risks associated with human and animal health.2020 Yıbar A, Soyutemiz E. Antibiotics use in food-producing animals and possible residual risk. Atatürk Üniversitesi Vet Bil Derg. 2013;8:97-104. Therefore, chicken meat, raw milk and cheese can be identified as high risk-carrying contributors for ESBL-producing Enterobacteriaceae and their encoding genes.99 Giedraitienė A, Vitkauskienė A, Naginienė R, Pavilonis A. Antibiotic resistance mechanisms of clinically important bacteria. Medicina (Kaunas). 2011;47:137-146.

The objective of this study was to determine the occurrence of ESBL-producing Enterobacteriaceae and the frequency rates of corresponding encoding genes in various foods of animal origin sold in Turkey.

Materials and methods

Reference cultures

An ESBL-negative strain Escherichia coli ATCC®25922 and an ESBL-positive strain Klebsiella pneumoniae ATCC®700603 were used as negative and positive controls, respectively, in the phenotypic and genotypic methods.

Sampling, sample preparation, isolation and identification of ESBL suspicious bacteria

During the period of May 2014 to November 2014, a total of 250 food samples of animal origin (100 packed raw chicken meat from public bazaars, markets and poultry farms, 100 raw cow milk from containers and holding tanks in bazaars and dairy farms, and 50 unpacked raw cow milk cheese from bazaars and markets) were randomly collected in different cities of Marmara, Aegean and Black Sea regions. All samples were placed into sampling bags, and taken to the laboratory in a thermobox container at 4 °C. The sample preparation and further examinations were quickly started after the sampling.

Ten millilitres of raw milk in 90 mL of Enterobacteriaceae Enrichment Broth (LABM, Lancashire, England), and 25 g either of raw cow milk, cheese or raw chicken meat in 225 mL of the mentioned broth was homogenised in a sterile bag (Interscience, Saint Nom, France) for 2 min by a stomacher (EasyMix, AES Chemunex, Rennes, France), and followed by an aerobic incubation at 37 °C for 18–24 h.2121 Geser N, Stephan R, Hächler H. Occurrence and characteristics of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk. BMC Vet Res. 2013;8:21 After that, 10 µL of the pre-enriched suspension was directly streaked onto Chromatic™ ESBL selective media (Liofilchem, Istanbul, Turkey). The inoculated plates were incubated at 37 °C for 18–24 h under aerobic condition as per to the manufacturer's instructions. The presumptive ESBL colonies were then sub-cultured onto Tryptic Soy Agar (LABM, Lancashire, England), followed by an incubation at 37 °C for 18–48 h. The species identification of suspected ESBL isolates was performed by Vitek® MS (bioMérieux, Marcy l’Etoile, France).

Screening and confirmation of ESBLs

Presumptive ESBL-producers were screened using cefpodoxime (CPD; 10 µg), cefotaxime (CTX; 30 µg), and ceftazidime (CAZ; 30 µg) antibiotic discs (MAST Group, UK). A 0.5 McFarland standard inoculum of the isolate was spread onto Mueller Hinton Agar (LABM, Lancashire, England) plates. The discs were inserted on the plate, and allowed for incubation at 37 °C for 18–24 h. The breakpoints with zone diameters were evaluated according to the criteria established by.2222 CLSI. Performance standards for antimicrobial susceptibility testing. Twenty-Third Informational Supplement. CLSI Document;2013. M100-S23. The ESBL Set D67C (MAST Group, UK) with a combination of CPD, CTX, and CAZ ± clavulanate (CLA, 10 µg) was used for disc diffusion confirmation test. The disc inserted plates were incubated at 37 °C for 18–24 h. The zone of inhibition was evaluated according to the criteria described by 2222 CLSI. Performance standards for antimicrobial susceptibility testing. Twenty-Third Informational Supplement. CLSI Document;2013. M100-S23..

Determination of minimal inhibitory concentration (MIC)

Micronaut-S beta-lactamase VII Plate (Merlin Diagnostika, Berlin, Germany) was used for the phenotypic detection of ESBL-production, including AmpC, metallo-beta-lactamase (MBL), and carbapenemase (KPC) according to the criteria described by 2222 CLSI. Performance standards for antimicrobial susceptibility testing. Twenty-Third Informational Supplement. CLSI Document;2013. M100-S23.. A 50 µL aliquot of 0.5 McFarland-standardised microbial suspension of the isolate was initially vortexed in 10 mL of Mueller Hinton Broth (Merck, Darmstadt, Germany). Subsequently, 100 µL of this suspension was pipetted into each well of the plate. After that, the plate was incubated overnight at 37 °C. The reading was recorded using a Thermofisher Multiskan FC Spectrometer. The MIC analysis was automatically performed by the MCN6 Software (Sifin, Berlin, Germany).

Extraction of bacterial DNA

Total DNA of ESBL-producing isolates was extracted using the GENESpin DNA Isolation Kit (Eurofins, Hamburg, Germany) according to the manufacturer's instructions. The extracted DNA samples were stored at –20 °C for further molecular analyses.

Characterisation of the bla variants

The primer pairs as previously described by 2323 Ojdana D, Sacha PB, Wieczorek P, et al. The occurrence of blaCTX-M, blaSHV, and blaTEM genes in extended-spectrum β-lactamase-positive strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland. Int J Antibiot. 2014;2014:935842 were used for screening of blaTEM, blaSHV and blaCTX-M. The blaCTX-M variants (blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, and blaCTX-M-25) were sub-characterised according to 2424 Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum b-lactamases. J Antimicrob Chemother. 2006;57:154-155.. All primers were prepared by Fullgen Biotechnology and İontek (İstanbul, Turkey). The amplification was performed by a Thermal Cycler PTC-0200G DNA Engine (BioRad, Istanbul, Turkey). The blaTEM, blaSHV and blaCTX-M were amplified in a singleplex PCR assay while a multiplex PCR assay was performed for blaCTX-M variants. The PCR mixtures were prepared in a final volume of 25 µL. All PCR conditions were optimised again as shown in Table 1. Gel-electrophoresis of the PCR products was performed on a 1.5% agarose-gel. The amplicons were photographed by GelDoc 2000 imaging system (BioRad, Istanbul, Turkey). Finally, the analysis was carried-out using Quantity One 4.6.3 GelDoc XR Software (BioRad, Istanbul, Turkey).

Table 1
Primer design, sequences, amplicon sizes and thermocycler conditions for bla-genes according to 2323 Ojdana D, Sacha PB, Wieczorek P, et al. The occurrence of blaCTX-M, blaSHV, and blaTEM genes in extended-spectrum β-lactamase-positive strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland. Int J Antibiot. 2014;2014:935842,2424 Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum b-lactamases. J Antimicrob Chemother. 2006;57:154-155..

Statistical analysis

The Kruskal–Wallis H -test was used for multiple comparison of the proportions of ESBL producers in their sampling groups. The test statistic H was approximated by a χ2 distribution. All statistical analyses were performed using SPSS 19 (SPSS Inc., USA) statistical package programme, and a P -value < 0.05 was considered as statistically significant.

Results

In this study, we screened, for ESBL-producing Enterobacteriaceae, 100 raw chicken meat samples, 100 raw cow milk samples, and 50 raw cow milk cheese samples from different cities of Marmara, Agean and Black Sea regions, and subsequently characterised their encoding blaTEM, blaSHV and blaCTX-M variants.

Phenotypic results

The phenotypic testing indicated that a total of 55 isolates (29 in raw chicken meat, 24 in raw cow milk, and 2 in raw cow milk cheese) from 250 food samples of animal origin were positive for ESBL-producing Enterobacteriaceae. The most prevalent phenotype was E. coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), K. pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) (Table 2).

Table 2
Screening results of the samples for ESBL-producing Enterobacteriaceae.

The disc approximation tests were performed by using CPD, CAZ, and CTX ± CLA containing discs according to the criteria set by 2222 CLSI. Performance standards for antimicrobial susceptibility testing. Twenty-Third Informational Supplement. CLSI Document;2013. M100-S23.. The average zone differences of CAZ ± CLA, CTX ± CLA and CPD ± CLA were found as 6.83 ± 3.66 mm, 11.97 ± 6.08 mm and 12.76 ± 6.06 mm in chicken meat, respectively, and 7.92 ± 5.07 mm, 13.33 ± 4.78 mm and 12.63 ± 7.11 mm in cow milk, respectively, while 10.00 ± 0.00 mm, 15.00 ± 4.24 mm and 14.50 ± 2.12 mm in cow milk cheese, respectively (Table 3).

Table 3
Zone diameters of CPD, CTX, and CAZ ± CLA.

The MIC determination revealed that 62.1% of the isolates from raw chicken meat were resistant to CTX, 55.2% to CAZ, 51.7% to CEP, 20.6% to COX, and 6.9% to CMC, while among the isolates from raw cow milk, 79.2% were resistant to CAZ, 75% to CTX, 66.6% to CEP, 54.2% to COX, 4.2% to ERT, and 4.2% to CMC. Two isolates from raw-cow-milk cheese were 100% resistant to CTX, CAZ, CEP, and CMC.

Among the 55 ESBL producers, five isolates (9.1%) were also positive for AmpC. Based on the species, the AmpC-producing isolates were E. coli (80%) and E. cloacae (20%) from raw chicken meat (n = 1), raw cow milk (n = 3), and raw-cow-milk cheese (n = 1). They were resistant to CAZ (100%), CTX and CEP (80%), CMC (40%), and COX (20%).

Genotypic results

In the genotypic analysis, the PCR products were identified: 53 as blaTEM, 36 as blaCTX-M, and 19 as blaSHV. In the 36 blaCTX-M, 35 were clustered as blaCTX-M-1, and one as blaCTX-M-8. The blaTEM was found in multiple combinations with blaCTX-M-1 & blaCTX-M-8 in 29, with blaSHV in 11, and with blaSHV & blaCTX-M-1 in 7. The blaSHV was detected in a combination with blaCTX-M-1 only in one instance. Of the 44 E. coli isolates, representing the most prevalent ESBL-producing phenotype, a co-existing distribution of bla genes was observed as blaTEM & blaCTX-M-1 in 25, blaSHV & blaTEM in six, blaSHV & blaTEM & blaCTX-M-1 in 6, blaSHV & blaCTX-M-1 in one, blaTEM & blaCTX-M-8 in one, blaTEM alone in four, and blaCTX-M-1 alone in one, respectively (Table 4).

Table 4
genotypic characteristics of 55 ESBL-producers isolated.

Statistical results

The multiple comparison of the proportions of ESBL producers in their sampling groups was performed using Kruskal–Wallis H -test (H = 5.935; P < 0.05). The test statistic H was approximated by χ2 distribution (CI 95%; χ2 = 12.5291; P = 0.001). Since H was less than χ2, no statistically significant difference was observed for the association between ESBL proportions and their sampling groups (P < 0.05) with respect to the food types of animal origin.

Discussion

In this study, we screened for ESBL-producing Enterobacteriaceae from raw chicken meat, raw cow milk and raw-cow-milk cheese samples. Subsequently, we characterised genotypically their encoding genes. The results highlighted that 55 isolates were positive for ESBL-producing Enterobacteriaceae; ESBL-producing E. coli (80%) was the most frequent species; a combination of blaTEM & blaCTX-M (52.7%) was the most common pattern; and the major variant of blaCTX-M was defined as blaCTX-M-1 (97.2%), respectively.

The ESBL encoding genetic elements are transferable between the same and different bacterial species.44 Rupp ME, Fey PD. Extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae: considerations for diagnosis, prevention and drug treatment. Drugs. 2003;63:353-365.,2525 Haque A, Yoshizumi A, Saga T, Ishii Y, Tateda K. ESBL-producing Enterobacteriaceae in environmental water in Dhaka, Bangladesh. J Infect Chemother. 2014;20:735-737. A study reported that the antibiotic residues in calf urine resulted in amplification of the populations of antibiotic resistant bacteria in the pen soil; and presumably, this increased the probability of transmission of these resistant populations to calves and then the animal-derived food chain.2626 Mitchell SM, Subbiah M, Ullman JL, Frear C, Call DR. Evaluation of 27 different biochars for potential sequestration of antibiotic residues in food animal production environments. J Environ Chem Eng. 2015;3:162-169. This might lead to a risk for infection and colonisation of the human intestinal microbiome with ESBL producing bacteria such as E. coli, E. cloacae, Klebsiella spp., Salmonella spp., Proteus spp. and Citrobacter spp., through various foods of animal origin2727 Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J. Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol. 2010;8:251-259.3030 Bonelli RR, Moreira BM, Picão RC. Antimicrobial resistance among Enterobacteriaceae in South America: history, current dissemination status and associated socioeconomic factors. Drug Resist Update. 2014;17:24-36.; when these foods are directly consumed without undergoing any thermal process, or used for raw milk cheese production.3131 Overdevest I, Willemsen I, Rijnsburger M, et al. Extended-spectrum beta-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands. Emerg Infect Dis. 2011;17:1216-1222. Due to this threat, the Codex Alimentarius Commission established an intergovernmental task force.3232 Doyle MP, Loneragan GH, Scott HM, Singer RS. Antimicrobial resistance: challenges and perspectives. Compr Rev Food Sci Foof Saf. 2013;12:234-248. Following that, in 2014, the Standard Committee of European Doctors (CPME), the Council of European Dentists (CED) and the Federation of Veterinarians of Europe (FVE) issued a common press release for all the authorities to manage the problem of ESBL-producing Enterobacteriaceae (www.fve.org). In our study, a high occurrence of ESBL producers and their encoding genes in the examined foods of animal origin indicate a health risk for the Turkish consumers.

Since the early 2000s, E. coli has become the most concerned member for infection and colonisation of the food animals.3333 Aidara-Kane A, Andremont A, Collignon P. Antimicrobial resistance in the food chain and the AGISAR initiative. J Infect Public Health. 2013;6:162-165.3636 Su Y, Yu CY, Tsai Y, Wang SH, Lee C, Chu C. Fluoroquinolone-resistant and extended-spectrum β-lactamase-producing Escherichia coli from the milk of cows with clinical mastitis in Southern Taiwan. J Microbiol Immunol Infect. 2014, http://dx.doi.org/10.1016/j.jmii.2014.10.003.
http://dx.doi.org/10.1016/j.jmii.2014.10...
An ESBL-producing E. coli associated mortality was three-times higher than non-ESBL producing E. coli.3737 Melzera M, Petersen I. Mortality following bacteraemic infection caused by extended spectrum beta-lactamase (ESBL) producing E. coli compared to non-ESBL producing E. coli. J Infect. 2007;55:254-259. The National Surveillance Network by the Ministry of Health in Turkey (www.uhes.saglik.gov.tr) has reported an increasing prevalence of ESBL-producing E. coli (33.2% in 2008 and 48.83% in 2013) and ESBL-producing K. pneumoniae (40% in 2008 and 49.69% in 2013). However, the role of foods in the dissemination of ESBL-producing bacteria and their encoding genes to humans has not been addressed so far in Turkey. In our study, the frequency of ESBL-producing E. coli was similar to those reported in Portugal (85%)3838 Amador P, Fernandes R, Prudêncio C, Brito L. Resistance to β-lactams in bacteria isolated from different types of Portuguese cheese. Int J Mol Sci. 2009;10:1538-1551. and Spain (93.3%),3333 Aidara-Kane A, Andremont A, Collignon P. Antimicrobial resistance in the food chain and the AGISAR initiative. J Infect Public Health. 2013;6:162-165. while it was higher than those reported in Denmark (1.3%),3939 Garcia-Migura L, Hendriksen RS, Fraile L, Aarestrup FM. Antimicrobial resistance of zoonotic and commensal bacteria in Europe: the missing link between consumption and resistance in veterinary medicine. Vet Microbiol. 2014;170:1-9. India (3.1%),4040 Kar D, Bandyopadhyay S, Bhattacharyya D, et al. Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India. Infect Genet Evol. 2015;29:82-90. China (12.4%),4141 Zheng H, Zeng Z, Chen S, et al. Prevalence and characterisation of CTX-M β-lactamases amongst Escherichia coli isolates from healthy food animals in China. Int J Antimicrob Agents. 2012;39:305-310. Germany (53.9%),4242 Schwaiger K, Huther S, Hölzel C, Kämpf P, Bauer J. Prevalence of antibiotic-resistant Enterobacteriaceae isolated from chicken and pork meat purchased at the slaughterhouse and at retail in Bavaria, Germany. Int J Food Microbiol. 2012;154:206-211. Belgium (45%),4343 Smet A, Martel A, Persoons D, et al. Diversity of extended-spectrum beta-lactamases and class C beta-lactamases among cloacal Escherichia coli isolates in Belgian broiler farms. Antimicrob Agents Chemother. 2008;52:1238-1243. Austria (24%)4444 Petternel C, Galler H, Zarfel G, et al. Isolation and characterization of multidrug-resistant bacteria from minced meat in Austria. Food Microbiol. 2014;44:41-46. and Taiwan (10.5%).3636 Su Y, Yu CY, Tsai Y, Wang SH, Lee C, Chu C. Fluoroquinolone-resistant and extended-spectrum β-lactamase-producing Escherichia coli from the milk of cows with clinical mastitis in Southern Taiwan. J Microbiol Immunol Infect. 2014, http://dx.doi.org/10.1016/j.jmii.2014.10.003.
http://dx.doi.org/10.1016/j.jmii.2014.10...
Our study also points E. coli being an important foodborne ESBL carrier.

Numerous phenotypic methods have been developed to detect ESBL production by Enterobacteriaceae. However, it is not still clear which tests are the most sensitive.4545 Garrec H, Drieux-Rouzet L, Golmard JL, Jarlier V, Robert J. Comparison of nine phenotypic methods for detection of extended-spectrum β-lactamase Production by Enterobacteriaceae. J Clin Microbiol. 2011;49:1048-1057. In our study, the phenotypic screening and confirmation of ESBL production were performed by the disc approximation tests. All of the used disc combinations showed good sensitivities in accordance with the criteria established by 2222 CLSI. Performance standards for antimicrobial susceptibility testing. Twenty-Third Informational Supplement. CLSI Document;2013. M100-S23.. After the disc approximation tests, MIC was determined due to the presence of ESBL encoding genes on the same plasmid.4646 Perez F, Endimiani A, Hujer KM, Bonomo RA. The continuing challenge of ESBLs. Curr Opin Pharmacol. 2007;7:459-469. The MIC revealed that 55 ESBL producers were resistant to CTX (MIC ≥ 4 µg/mL) and CAZ (MIC ≥ 16 µg/mL), and partly variable to CPD (MIC ≥ 8 µg/mL). Basically, TEM and SHV type beta-lactamases are resistant to CAZ, and variable to CTX while CTX-M type beta-lactamase exhibits resistance to CPD and variable to CAZ because CTX-M type beta-lactamase may escape from screening by CAZ. A survey in Europe reported that up to 33% of ESBLs strains remained undetected, therefore, in this case, genotypic confirmation is further needed.4747 Naas T, Oxacelay C, Nordmann P. Identification of CTX-M-Type extended-spectrum-β-lactamase genes using real-time PCR and pyrosequencing. Antimicrob Agents Chemother. 2007;51:223-230.5050 Taneja N, Sharma M. ESBL detection in clinical microbiology: why & how?. Indian J Med Res. 2008;127:297-300. In our study, the phenotypic results indicated that TEM and CTX-M type beta lactamases were the most commonly encountered ESBLs.

The co-existence of ESBL and AmpC beta-lactamases is a worldwide growing concern.55 EFSA. Scientific opinion on the public health risks ofbacterial strains producing extended-spectrum β-lactamasesand/or AmpC β-lactamases in food and food-producinganimals. EFSA J. 2011;9:2322–2417.,5151 Thomson KS. Extended-spectrum-β-lactamase, AmpC, and carbapenemase issues. J Clin Microbiol. 2010;48:1019-1025. ESBL and AmpC producing enterobacteria have been clinically reported as 19.5% in Europe, and as 13.9% in Turkey.5252 Turner PJ. MYSTIC Europe 2007: activity of meropenem and other broad-spectrum agents against nosocomial isolates. Diagn Microbiol Infect Dis. 2009;63:217-222.,5353 Korten V, Ulusoy S, Zarakolu P, Mete B. Antibiotic resistance surveillance over a 4-year period (2000–2003) in Turkey: results of the MYSTIC program. Diagn Microbiol Infect Dis. 2007;59:453-457. In our study, AmpC producers were E. coli and E. cloacae, as reported earlier.5252 Turner PJ. MYSTIC Europe 2007: activity of meropenem and other broad-spectrum agents against nosocomial isolates. Diagn Microbiol Infect Dis. 2009;63:217-222.,5454 Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 2002;40:2153-2162.,5555 Kiratisin P, Henprasert A. Genotypic analysis of plasmid-mediated beta-lactamases amongst Enterobacteriaceae other than Escherichia spp. and Klebsiella spp. that are non-susceptible to a broad-spectrum cephalosporin. Int J Antimicrob Agents. 2010;36:343-347. Failure to detect these beta lactamases has contributed to their uncontrolled spread and sometimes the consequent therapeutic failures.5656 Thomson KS. Controversies about extended-spectrum and AmpC beta-lactamases. Emerg Infect Dis. 2001;7:333-336. For Enterobacteriaceae, the use of either cefepime (CEP) or cefpirome (CPO) ± CLA is recommended since they are un-affected by AmpC.5151 Thomson KS. Extended-spectrum-β-lactamase, AmpC, and carbapenemase issues. J Clin Microbiol. 2010;48:1019-1025. In contrast, our results revealed that AmpC producers were 100% resistant to CEP + CLA. This indicates that the techniques to detect AmpC beta-lactamase are still evolving and are not yet optimised.5757 Jacoby GA. AmpC beta-lactamases. Clin Microbiol Rev. 2009;22:161-182.

The genotypic characterisation by PCR assays revealed that blaTEM was the most frequent gene, followed by blaCTX-M and blaSHV. In Turkey, the clinically reported most frequent beta-lactamase type was CTX-M, followed by TEM and SHV, while another study reported a frequency of SHV higher than TEM.1515 Sharma M, Pathak S, Srivastava P. Prevalence and antibiogram of extended spectrum β-lactamase (ESBL) producing Gram negative bacilli and further molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. J Clin Diagn Res. 2013;7:2173-2177.,5858 Nazik H, Bektore B, Ongen B, et al. Plasmid-mediated quinolone resistance genes in Escherichia coli urinary isolates from two teaching hospitals in Turkey: coexistence of TEM, SHV, CTX-M and VEB-1 Type β-lactamases. Trop J Pharm Res. 2011;10:325-333.,5959 Zaniani FR, Meshkat Z, Nasab MN, et al. The prevalence of TEM and SHV genes among extended-spectrum betalactamases producing Escherichia coli and Klebsiella pneumoniae. Iran J Basic Med Sci. 2012;15:654-660. Our genotypic results reporting the most frequent genes as blaTEM & blaCTX-M were also epidemiologically similar to the most commonly encountered ESBL encoding genes in chicken meat from Eastern Europe and Near East,77 Bonnet R. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004;48:1-14. Southern America,3030 Bonelli RR, Moreira BM, Picão RC. Antimicrobial resistance among Enterobacteriaceae in South America: history, current dissemination status and associated socioeconomic factors. Drug Resist Update. 2014;17:24-36.,6060 Ferreira JC, Filho RACP, Andrade LN, Junior AB, Darini ALC. IncI1/ST113 and IncI1/ST114 conjugative plasmids carrying blaCTX-M-8 in Escherichia coli isolated from poultry in Brazil. Diagn Microbiol Infect Dis. 2014;80:304-306. Tunisia,6161 Chouchani C, Marrakchi R, El Salabi A. Evolution of β-lactams resistance in Gram-negative bacteria in Tunisia. Crit Rev Microbiol. 2011;37:167-177. Northern European countries,3333 Aidara-Kane A, Andremont A, Collignon P. Antimicrobial resistance in the food chain and the AGISAR initiative. J Infect Public Health. 2013;6:162-165. Japan,6262 Hiroi M, Yamazaki F, Harada T, et al. Prevalence of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals. J Vet Med Sci. 2012;74:189-195.,6363 Kawamura K, Goto K, Nakane K, Arakawa Y. Molecular epidemiology of extended-spectrum β-lactamases and Escherichia coli isolated from retail foods including chicken meat in Japan. Foodborne Pathog Dis. 2014;11:104-110. Germany,6464 Reich F, Atanassova V, Klein G. Extended-spectrum β-lactamase- and AmpC-producing enterobacteria in healthy broiler chickens, Germany. Emerg Infect Dis. 2013;19:1253-1259. China,3535 Rao L, Lv L, Zeng Z, et al. Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003–2012. Vet Microbiol. 2014;172:534-541. Austria6565 Zarfel G, Galler H, Luxner J, et al. multiresistant bacteria isolated from chicken meat in Austria. Int J Environ Res Public Health. 2014;11:12582-12593. and India,4040 Kar D, Bandyopadhyay S, Bhattacharyya D, et al. Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India. Infect Genet Evol. 2015;29:82-90. followed by the raw milk from Spain,2121 Geser N, Stephan R, Hächler H. Occurrence and characteristics of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk. BMC Vet Res. 2013;8:21 England6666 Randall L, Heinrich K, Horton R, et al. Detection of antibiotic residues and association of cefquinome residues with the occurrence of extended-spectrum β-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011. Res Vet Sci. 2014;96:15-24. and Taiwan3636 Su Y, Yu CY, Tsai Y, Wang SH, Lee C, Chu C. Fluoroquinolone-resistant and extended-spectrum β-lactamase-producing Escherichia coli from the milk of cows with clinical mastitis in Southern Taiwan. J Microbiol Immunol Infect. 2014, http://dx.doi.org/10.1016/j.jmii.2014.10.003.
http://dx.doi.org/10.1016/j.jmii.2014.10...
, and by the cheese and dairy products from Portugal,3838 Amador P, Fernandes R, Prudêncio C, Brito L. Resistance to β-lactams in bacteria isolated from different types of Portuguese cheese. Int J Mol Sci. 2009;10:1538-1551. England,6666 Randall L, Heinrich K, Horton R, et al. Detection of antibiotic residues and association of cefquinome residues with the occurrence of extended-spectrum β-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011. Res Vet Sci. 2014;96:15-24. Iran6767 Khoshbakht R, Shahed A, Aski HS. Characterization of extended spectrum B-lactamase-producing Escherichiae coli srains isolated from dairy products. J Microbiol Biotechnol Food Sci. 2014;3:333-336. and Egypt,6868 Ahmed AM, Shimamoto T. Molecular analysis of multidrug resistance in Shiga toxin-producing Escherichia coli O157:H7 isolated from meat and dairy products. Int J Food Microbiol. 2015;193:68-73. respectively. However, the foodborne ESBL-producing bacteria and their encoding genes in Turkey have not been addressed till date, and the foodborne beta-lactamases have been studied only by disc approximation testing, excluding genotypic characterisation.6969 Gundogan N, Yakar U. Siderophore production, serum resistance, hemolytic activity and extended spectrum beta lactamase producing Klebsiella species isolated from milk and milk products. J Food Saf. 2007;3:251-260.7171 Gundogan N, Avci E. Prevalence and antibiotic resistance of extended spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella species isolated from foods of animal origin in Turkey. Afr J Microbiol Res. 2013;7:4059-4064. Therefore, our study confirms that the foods of animal origin in Turkey are potential reservoirs of diverse ESBL encoding genes with a co-resistance pattern, which can be disseminated to clinical and community settings.

We detected CTX-M-1 cluster as the most prevalent sub-type of CTX-M type beta lactamases. Chromosomal bla gene, kluC, present in Kluyvera cryocrescens is the ancestor of CTX-M-1 cluster.7272 Cantón R, González-Alba JM, Galán JC. CTX-M enzymes: origin and diffusion. Front Microbiol. 2012;3:110 The CTX-M type ESBLs are currently recognised as the part of the most threatening mechanism of antibiotic resistance in clinical and community settings, virtually invading all human and animal compartments as well as the environment all over the world, and are increasingly being predominant form in Enterobacteriaceae worldwide.4747 Naas T, Oxacelay C, Nordmann P. Identification of CTX-M-Type extended-spectrum-β-lactamase genes using real-time PCR and pyrosequencing. Antimicrob Agents Chemother. 2007;51:223-230.,7272 Cantón R, González-Alba JM, Galán JC. CTX-M enzymes: origin and diffusion. Front Microbiol. 2012;3:1107474 Hawkey PM. Multidrug-resistant Gram-negative bacteria: a product of globalization. J Hosp Infect. 2015;89:241-247. South America is known as an important source of CTX-M type ESBL originated from clinical settings.7575 Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-type extended-spectrum β-lactamases. Clin Microbiol Infect. 2008;14:33-41. In Turkey, CTX-M-1 group was found in 86.8% of the clinical E. coli isolates.7676 Gonullu N, Aktas Z, Kayacan CB, et al. Dissemination of CTX-M-15 β-lactamase genes carried on Inc FI and FII plasmids among clinical isolates of Escherichia coli in a University Hospital in Istanbul, Turkey. J Clin Microbiol. 2008;46:1110-1112. Our results showed that the dissemination of CTX-M-1 cluster was not restricted to the clinical settings, but also involved the foods of animal origin, causing rapid, important and unpredictable changes in the epidemiology of antibiotic resistance.7575 Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-type extended-spectrum β-lactamases. Clin Microbiol Infect. 2008;14:33-41.

In 2007, European Food Safety Authority (EFSA) recommended that bacterial strains containing transferable resistance genes should not be used in animal feeds and fermented and probiotic foods for human use.7777 Vankerckhoven V, Huys G, Vancanneyt M, et al. Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE Project. Trends Food Sci Technol. 2008;19:102-114.,7878 Sharma P, Tomar SK, Goswami P, Sangwan V, Singh R. Antibiotic resistance among commercially available probiotics. Food Res Int. 2014;57:176-195. However, the issue of antibiotics resistance bacteria in foods has not yet been seriously raised as a health safety indicator. In conclusion, the foods of animal origin sold in Turkey have been found as potential reservoirs for diverse ESBL-producing Enterobacteriaceae and their encoding genes with a co-resistance pattern, thus posing a critical health risk for the Turkish consumers.

Disclaimer

This article is based on the Doctoral thesis entitled “Characterization of Extended Spectrum Beta-Lactamases in Enterobacteriaceae from Foods Using Molecular Method” by İsmail Hakkı Tekiner, 2015, Department of Food Engineering, Institute of Natural and Applied Sciences, Istanbul Aydın University, Istanbul, Turkey.

  • Associate Editor: Ana Lúcia da Costa Darini

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Publication Dates

  • Publication in this collection
    Apr-Jun 2016

History

  • Received
    16 Apr 2015
  • Accepted
    12 Nov 2015
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br