SciELO - Scientific Electronic Library Online

 
vol.47 número7Acidentes com aeronaves especializadas em aplicações aéreas na agriculturaFrequência e fatores de riscos associados a caprinos soropositivos a Leptospiras spp. no Estado de Sergipe, Nordeste do Brasil índice de autoresíndice de assuntospesquisa de artigos
Home Pagelista alfabética de periódicos  

Serviços Personalizados

Journal

Artigo

Indicadores

Links relacionados

Compartilhar


Ciência Rural

versão On-line ISSN 1678-4596

Cienc. Rural vol.47 no.7 Santa Maria  2017  Epub 29-Maio-2017

http://dx.doi.org/10.1590/0103-8478cr20170008 

MICROBIOLOGY

Methicillin-resistant Staphylococcus spp. isolated from curd cheese “requeijão” and “especialidade láctea type requeijão” sold in Brazil

Isolamento de Staphylococcus spp. resistente à meticilina de queijos fundidos requeijão e especialidade láctea tipo requeijão comercializados no Brasil

Jamile de Oliveira Hachiya1 

Gabriel Augusto Marques Rossi1  * 

Laryssa Freitas Ribeiro1 

Rafael Akira Sato1 

Higor Oliveira Silva1 

Ana Maria Centola Vidal2 

Luiz Augusto do Amaral1 

1Departamento de Medicina Veterinária Preventiva e Reprodução Animal, Faculdade de Ciências Agrárias e Veterinárias (FCAV), Universidade Estadual Paulista (UNESP), Via de Acesso Paulo Castellane, s/n, 14884-900, Jaboticabal, SP, Brasil.

2Departamento de Medicina Veterinária, Universidade de São Paulo (UNESP), Faculdade de Zootecnia e Engenharia de Alimentos (FZEA), Pirassununga, SP, Brasil.

ABSTRACT:

This study focused on counting Staphylococcus spp. in curd cheeses “requeijão” and “especialidade láctea type requeijão” sold in Brazil, assessing the presence of mecA gene in obtained isolates and establishing antimicrobial resistance profile of the mecA gene positive isolates. To this, a set of 200 samples of these dairy products were evaluated. Low counts of Staphylococcus spp. were observed in these dairy products. All the isolates were determined as coagulase-negative strains using coagulase test and PCR. However, two isolates (3.70%) were carriers of mecA gene and they can be considered as risk for public health. These isolates presented resistance to penicillin, oxacillin and erythromycin. In conclusion, low counts of Staphylococcus were detected in curd cheese “requeijão” and “especialidade láctea type requeijão” sold in Brazil. However, coagulase-negative methicillin-resistant Staphylococcus spp. was detected in these dairy products. This is the first report of the detection of methicillin-resistant Staphylococcus spp. in heat-treated dairy products in Brazil. Results served as a warning to public sanitary authorities to control multidrug-resistant strains in veterinary and human medicine.

Key words: antimicrobial resistance; cheese; food safety; MRS; Staphylococcus spp

RESUMO:

Este trabalho objetivou realizar a contagem de Staphylococcus spp. em queijos fundidos “requeijão” e “especialidade láctea tipo requeijão” comercializados no Brasil, verificar a presença do gene mecA nos isolados obtidos e estabelecer o perfil de resistência antimicrobiana dos isolados positivos para tal gene. Para isso, 200 amostras desses produtos lácteos foram avaliadas. Baixas contagens de Staphylococcus spp. foram observadas nas amostras. Todos os isolados foram considerados como coagulase-negativos através do teste da coagulase e através da PCR. Entretanto, em dois isolados (3,70%) foi possível detectar o gene mecA e representam potencial risco à saúde pública. Esses isolados apresentaram resistência a penicilina, oxacilina e eritromicina. Conclui-se que baixas contagens de Staphylococcus foram detectadas em queijos fundidos “requeijão” e “especialidade láctea type requeijão”. Entretanto, cepas de Staphylococcus spp. coagulase-negativas e resistentes à meticilina foram detectadas nesses derivados lácteos. Esse é o primeiro relato da ocorrência de cepas de Staphylococcus spp. resistentes à meticilina em produtos lácteos termicamente tratados comercializados no Brasil. Os resultados servem como um alerta para as autoridades sanitárias públicas nacionais para o controle de cepas multirresistentes em medicina veterinária e humana.

Palavras-chave: resistência antimicrobiana; segurança alimentar; MRS; queijos; Staphylococcus spp

INTRODUCTION:

“Requeijão” is the third most sold cheese in Brazil (ZACARCHENCO et al., 2012). “Requeijão” is defined as “the product obtained using acid or enzymatic milk coagulation followed by the curd mass fusion (80ºC for 5 seconds), whey removal and washing according to Brazilian legislation (BRAZIL, 1997). Also, addition of milk cream, butter or butter oil in its composition is allowed. Another curd cheese routinely consumed in Brazil is “especialidade láctea type requeijão”, a similar cheese produced using the same process but the addition of vegetable ingredients such as corn starch and vegetable oil is allowed in composition.

The genus Staphylococcus is among the most important pathogenic bacteria reported in dairy products. This genus is divided into two groups: coagulase-positive and coagulase-negative strains. Among the first one, S. aureus is known for its clinical importance and as a foodborne pathogen. The coagulase-negative group is comprised by several species involved in opportunistic infections in immunocompromised patients (MARTINS & CUNHA, 2007).

Staphylococcus spp. are also frequently associated to mastitis (PITKALA et al., 2004), biofilm formation (OTTO, 2009) and are closely related to improper food handling of food handlers (ARGUDÍN et al., 2012), leading contamination of milk and other dairy products, posing public health into risk. Consequently, Staphylococcus spp. antimicrobial resistance is a concern for public health (KOKSAL et al., 2009).

Methicillin resistance is an increasing global problem (MARTINS & CUNHA, 2007), even for domestic animals (CUNY et al, 2010). The presence of these isolates in food and animals highlight the potential risk for human transmission (WEESE & DUJIKEREN, 2010). Some researchers have reported strains of methicillin resistant Staphylococcus spp. (MRS) causing mastitis (MOON et al., 2007), in bulk tank milk (VIRGIN et al., 2009), in bovine milk, pecorino and mozzarella cheeses (NORMANO et al., 2009) and in raw meats (BOER et al., 2009). Based on all these information, this study focused on counting Staphylococcus spp. in curd cheeses “requeijão” and “especialidade láctea type requeijão” sold in Brazil, assessing the presence of mecA gene in obtained isolates and establishing antimicrobial resistance profile of the mecA gene positive isolates.

MATERIALS AND METHODS:

This research was performed using samples of “requeijão”, “requeijão light”, “especialidade láctea type requeijão” and “especialidade láctea type requeijão light”, obtained from supermarkets located in the municipality of Jaboticabal, state of São Paulo, Brazil, during the year of 2015. Samples were kept refrigerated in cooler boxes with ice, during the transport to laboratory. Each kind of dairy product (“requeijão”, “requeijão light”, “especialidade láctea type requeijão”) had 50 samples bought, from 5 different commercials brands, each one with 2 different production lots (each lot composed by 5 samples). For the “especialidade láctea type requeijão light”, the samples were from three different commercial brands and 10 different production lots (each lot composed by 5 samples). In total, 200 samples were used for this study.

For each sample, 25g was placed into a Scott Bottle containing 225mL of 0.1% peptone water (Difco, Detroit, USA). Out of this first dilution (10-1), other serial dilutions were performed, until 10-3. Selective plating was done using 0.1mL of these dilutions in Baird-Parker agar (Difco, Detroit, USA). Plates were incubated for 45-48 hours under 35ºC. Whenever black colonies grew in the agar, it was transferred to a tube with Tryptic Soy Agar (TSA) (Difco, Detroit, USA) medium, and incubated for 24 hours under 35ºC (APHA, 2011). Moreover, the colonies were transferred to tubes containing Brain Heart Infusion broth (BHI) (Difco, Detroit, USA) and incubated for 24 hours under 37ºC. The coagulase test was performed using the isolates from BHI broth and rabbit plasma, in order to assess the presence of coagulase-positive strains (APHA, 2011).

The DNA from the 57 isolates was extracted according to the method described by KESKIMAKI et al. (2001). Presence of Staphylococcus spp. and S. aureus DNA molecules was detected using primers 16SrRNA (Pereira et al., 2009) and Sa442-1/2 (MOROT-BIZOT et al., 2004). A PCR also was performed to identify coagulase-positive Staphylococcus spp. (S. intermedius, S. schleiferi subsp. coagulans, S. hyicus, S. lutrae, S. delphini, and S. pseudintermedius according to SASAKI et al. (2010) (Table 1). In order to detect methicillin-resistant isolates (mecA), a PCR was performed using the primers as described by GORTEL et al. (1999): 5’ AAA ATC GAT GGT AAA GGT TGG C 3’ e 5’ AGT TCT GCA GTA CCG GAT TTG C 3’, with a 533bp product and 55°C annealing temperature.

Table 1 Primer, nucleotides sequence, amplicon size (bp), annealing temperature, positive control and reference of each primer used for the detection of Staphylococcus spp. 

PCR amplifications were carried out with mixtures containing PCR buffer 1x [100mM Tris-HCl pH 8.8; 500mMKCl; 0.8% (v/v) Nonidet P40]; MgCl2 2mM; dNTP’s 0.2mM, 1.5U de Taq DNA polymerase, 5pmol of each primer, 60ng genomic DNA and sterile water until reaching the volume of 20µL. Cycling conditions consisted of an initial step at 95°C for 5min, followed by 35 cycles at 94ºC for 1min, annealing at the specific temperature of each primer for 1min and 72ºC for 1.5min. The final extension was at 72°C for 10min. The amplified products were visualized in a 1% agarose gel electrophoresis stained with 0.5µg mL-1 ethidium bromide (Vetec, São Paulo, Brazil). A previously known molecular weight marker was used as a reference (GeneRuler 1kb DNA Ladder - Fermentas, Thermo Fish Scientific, São Paulo, Brazil).

MRS positive isolates were examined for resistance to 11 antimicrobials by disk-diffusion (Kirby-Bauer) method. The following disks (LaborclinTM) were used: oxacillin (1µg), cefepime (30µg), rifampicin (5µg), chloramphenicol (30µg), clindamyicin (2µg), erythromycin (15µg), penicillin G (10un), sulfatrim (23,75/1,25μg), tetracycline (30µg), gentamicin (10µg) and ciprofloxacin (5µg).

The significance between the Staphylococcus spp. counting of the groups was assessed through a variance analysis and the Kruskall-Wallis test with 5% of significance level. Data regarding the “presence or absence” of Staphylococcus spp. in the samples group (comparison among light and normal products; and among “requeijão” and “especialidade láctea” products) was performed using the chi-square test with 5% of significance level. The analyses were performed using Software R®.

RESULTS AND DISCUSSION:

Table 2 presents Staphylococcus spp. counts of the dairy product samples analyzed in this study. Results of Staphylococcus spp. count did not meet the necessary requirements to perform the variance analysis. Moreover, when using the nonparametric Kruskal-Wallis test with 5% of significance level, no statistical significance (P>0.05) was observed among these products. The chi-square test performed to evaluate the difference among the presence or absence of these microorganisms in the products also presented no significance (P>0.05).

Table 2 Average Staphylococus spp. count in “requeijão”, “requeijão light”, “especialidade láctea type requeijão” and “especialidade láctea type “requeijão light” sold in Jaboticabal municipality, state of São Paulo, Brazil during 2015. 

Staphylococcus spp. count in the samples was low and all the strains were considered as coagulase-negative strains on coagulase-test. S. aureus is considered as the most important foodborne pathogen of this group and it is considered as coagulase-positive specie. Based on such results, it is possible to state that the consumption of these dairy products does not directly pose risk to the Brazilian population, with regard to foodborne illness occurrence. Furthermore, the low number of the pathogen observed in the samples could be explained by the high temperature processes used in the production flowchart (BRAZIL, 1997) and good manufacturing practices implementation in these industries (SANTANA et al., 2009).

A set of 57 isolates were subjected to a PCR in order to confirm if they were Staphylococcus spp. (PEREIRA et al., 2009), S. aureus (MOROT-BIZOT et al., 2004) or other coagulase-positive Staphylococcus spp. (S. intermedius, S. schleiferi subsp. coagulans, S. hyicus, S. lutrae, S. delphini, and S. pseudintermedius (SASAKI et al., 2010). In total 54 isolates were confirmed as Staphylococcus spp. with amplification of the corresponding product. There were no amplification products using primers for Staphylococcus spp. coagulase-positive, agreeing with the coagulase-test results and allowing us to confirm that all isolates were coagulase-negative strains.

Using PCR to detect the presence of mecA gene, one isolate obtained from “requeijão light” (brand C and lot 2) and another isolate obtained from “especialidade láctea type requeijão light” (brand C and lot 2) were considered as positive. The prevalence of methicillin-resistant coagulase-negative Staphylococcus spp. in evaluated samples was 3.70% among the 54 isolates. This two isolates showed resistance to oxacillin, penicillin G and erythromycin but did not to cefepime, rifampicin, chloramphenicol, clindamyicin, sulfatrim, tetracycline, gentamicin and ciprofloxacin antimicrobials. Resistance to certain non-beta-lactam (as erythromycin) in Staphylococcus spp. isolates carrying mecA gene was already reported (HUBER et al., 2011).

NORMANNO et al. (2007) established 3.75% as the prevalence of methicillin-resistant Staphylococcus spp. in milk and dairy products in Italy highlighting the role of dairy products in transmission of antimicrobial-resistant bacteria to humans. Furthermore, some authors isolated strains of Staphylococcus spp. methicillin-resistant from some food such as: cow’s milk, pecorino and mozzarella cheeses (NORMANO et al., 2009) and raw meat (BOER et al., 2009). However, the occurrence of methicillin-resistant Staphylococcus spp. in “requeijão” and “especialidade láctea”, or even in any other heat-treated dairy product, was never reported in Brazil, until this paper.

“Requeijão” is one of the most consumed cheeses in Brazil and it is usually consumed raw (ZACARCHENCO et al., 2012). Presence of coagulase-negative methicillin-resistant Staphylococcus spp. strains in these products present a risk to the country’s public health. Thus, considering the potential implications in food production chain, there is a need to be constantly monitoring the employers’ health and hygiene, once humans can be colonized by contact during food processing or consumption of contaminated food (KLUYTMANS, 2009).

Moreover, nowadays coagulase-negative Staphylococcus spp. is considered as able on transferring antimicrobial resistance genes to other Staphylococcus spp. strains (TULINSKI et al., 2012). Human infections caused by methicillin-resistant Staphylococcus spp. are increasing (MARTINS & CUNHA, 2007), even in animals (CUNY et al., 2010), being considered an emergent problem in veterinary medicine and public health (HUBER et al., 2011). Animals usually are infected and colonized during contact with humans and vice-versa (GRAVELAND et al., 2010). Consequently, prophylactic measures to avoid methicillin-resistant Staphylococcus spp. contamination in foods and infections in humans and animals are required in order to reduce risk in Brazil.

CONCLUSION:

In conclusion, processing technology ensures the safety of these products categories; however, they may be carriers of methicillin resistant Staphylococcus spp. strains. Results served as a warning to Brazilian public sanitary authorities to control multidrug-resistant strains in veterinary and human medicine

ACKNOWLEDGMENTS

The authors acknowledge the financial support of this research by the Brazilian research funding agency Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Process no. 14/21534-6)

REFERENCES:

APHA (AMERICAN PUBLIC HEALTH ASSOCIATION). Committee on Microbiological for Foods. Compendium of methods for the microbiological examination of foods. 4.ed. Washington, 2011. 676p. [ Links ]

ARGUDÍN, M.A et al. Genotypes, exotoxin gene content and antimicrobial resistance in Staphylococcus aureus recovered from food and food-handlers. Applied and Environmental Microbiology, v.78, n.8, p.2930-2935, 2012. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318794/ >. Accessed: Jan. 04, 2017. doi: 10.1128/AEM.07487-11. [ Links ]

BOER, E. et al. Prevalence of methicillin-resistant Staphylococcus aureus in meat. International Journal of Food Microbiology, v.134, n.1-2, p.52-56, 2009. Available from: http://www.sciencedirect.com/science/article/pii/S0168160508006466 >. Accessed: Dec. 29, 2016. doi: 10.1016/j.ijfoodmicro.2008.12.007. [ Links ]

BRAZIL. Ministry of Supply, Agriculture and Livestock. Portaria nº 359, de 4 de setembro de 1997. Official Gazzete, Brasília, DF, Sept. 08, 1997. Section 1, p.19690-19691. [ Links ]

CUNY C. et al. Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species. International Journal of Medical Microbiology, v.300, n.2-3, p.109-117, 2010. Available from: http://www.sciencedirect.com/science/article/pii/S1438422109001313 >. Accessed: Dec. 28, 2017. doi: 10.1016/j.ijmm.2009.11.002. [ Links ]

GORTEL, K. et al. Methicillin resistance among staphylococci isolated from dogs. American Journal of Veterinary Research, v.60, n.12, p.1526-1530, 1999. Available from: https://www.ncbi.nlm.nih.gov/pubmed/10622162 >. Accessed: Jan. 04, 2017. [ Links ]

GRAVELAND, H. et al. Methicillin resistantStaphylococcus aureusST398 in veal calf farming: human MRSA carriage related with animal antimicrobial usage and farm hygiene. Plos One, v.5, n.6, p.e10990, 2010. Available from: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010990 >. Accessed: Jan. 03, 2017. doi: 10.1371/journal.pone.0010990. [ Links ]

HUBER, H. et al. Prevalence and characteristics of methicillin resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons. BMC Veterinary Research, v.7, n.6, p.1-7, 2011. Available from: http://bmcvetres.biomedcentral.com/articles/10.1186/1746-6148-7-6 >. Accessed: Dec. 06, 2016. doi: 10.1186/1746-6148-7-6. [ Links ]

KESKIMAKI, M. et al. EPEC, EAEC and STEC in stool specimens: prevalence and molecular epidemiology of isolates. Diagnostic Microbiology & Infectious Disease, v.40, n.4, p.151-156, 2001. Available from: http://www.dmidjournal.com/article/S0732-8893(01)00265-6/abstract >. Accessed: Jan. 04, 2017. doi: 10.1016/S0732-8893(01)00265-6. [ Links ]

KLUYTMANS, J.A.J.W. Methicillin-resistant Staphylococcus aureus in food products: cause for concern or case for complacency? Clinical Microbiology & Infection, v.16, n.1, p.11-15, 2009. Available from: http://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)617778/abstract >. Accessed: Jan. 01, 2017. doi: 10.1111/j.1469-0691.2009.03110.x. [ Links ]

KOKSAL, F. et al. Antibiotic resistance patterns of coagulase-negative Staphylococcus strains isolated from blood cultures of septicemic patients in Turkey. Microbiological Research, v.164, n.4, p.404-410, 2009. Available from: http://www.sciencedirect.com/science/article/pii/S0944501307000560 >. Accessed: Jan. 03, 2017. doi: 10.1016/j.micres.2007.03.004. [ Links ]

MARTINS, A.; CUNHA, M.L.R.S. Methicillin resistance in Staphylococcus aureus and Coagulase-Negative Staphylococci: epidemiological and molecular aspects. Microbiology & Immunology, v.51, n.9, p.787-795, 2007. Available from: https://www.ncbi.nlm.nih.gov/pubmed/17895595 >. Accessed: Dec. 28, 2017. doi: 10.1111/j.1348-0421.2007.tb03968.x. [ Links ]

MOON, J.S. et al. Phenotypic and genetic antibiogram of Methicillin-resistant Staphylococci isolated from bovine mastitis in Korea. Journal of Dairy Science, v.90, n.3, p.1176-1185, 2007. Available from: https://www.ncbi.nlm.nih.gov/pubmed/17297092 >. Accessed: Jan. 04, 2017. doi: 10.3168/jds.S0022-0302(07)71604-1. [ Links ]

MOROT-BIZOT, S.C. et al. Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food. Journal of Appied Microbiology, v.97, n.5, p.1087-1094, 2004. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2004.02399.x/abstract >. Accessed: Jan. 04, 2017. doi: 10.1111/j.1365-2672.2004.02399.x. [ Links ]

NORMANNO, G. et al. Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy. International Journal of Food Microbiology , v.117, n.2, p.219-222, 2007. Available from: http://www.sciencedirect.com/science/article/pii/S0168160507002565 >. Accessed: Dec. 22, 2016. doi: 10.1016/j.ijfoodmicro.2007.04.006. [ Links ]

OTTO, M. Staphylococcal biofilms. Current Topics in Microbiology and Immunology, v.322, p.207-228, 2009. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777538/ >. Accessed: Jan. 04, 2017. [ Links ]

PEREIRA, V. et al. Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various food in Portugal. Food Microbiology, v.26, n.3, p.278-282, 2009. Available from: https://www.ncbi.nlm.nih.gov/pubmed/19269569 >. Accessed: Jan. 04, 2017. doi: 10.1016/j.fm.2008.12.008. [ Links ]

PITKALA, A. et al. Bovine mastitis in Finland 2001 - Prevalence, distribution of bacteria, and antimicrobial resistance. Journal of Dairy Science , v.87, n.8, p.2433-2441, 2004. Available from: https://www.ncbi.nlm.nih.gov/pubmed/15328265 >. Accessed: Jan. 02, 2017. doi: 10.3168/jds.S0022-0302(04)73366-4. [ Links ]

SANTANA, N.G. et al. Microbiological quality and safety of meals served to children and adoption of good manufacturing practices in public school catering in Brazil. Food Control v.20, n.3, p.255-261, 2009. Available from: http://www.sciencedirect.com/science/article/pii/S0956713508001217 >. Accessed: Jan. 03, 2017. doi: 10.1016/j.foodcont.2008.05.004. [ Links ]

SASAKI, T. et al. Multiplex-PCR method for species identification of coagulase-positive Staphylococci. Journal of Clinical Microbiology, v.48, n.3, p.765-769, 2010. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832457/ >. Accessed: Dec. 29, 2016. doi: 10.1128/JCM.01232-09. [ Links ]

TULINSKI, P. et al. Methicillin-resistant coagulase-negative Staphylococci on pig farms as a reservoir of heterogeneous Staphylococcal cassette chromosome mec elements. Applied and Environmental Microbiology , v.78, n.2, p.299-304, 2012. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255757/ >. Accessed: Jan. 04, 2017. doi: 10.1128/AEM.05594-11. [ Links ]

VIRGIN, J.E. et al. Short communication: Methicillin-resistant Staphylococcus aureus detection in US bulk tank milk. Journal of Dairy Science , v.92, n.10, p.4988-4991, 2009. Available from: http://www.sciencedirect.com/science/article/pii/S0022030209708306 >. Accessed: Jan. 03, 2017. doi: 10.3168/jds.2009-2290. [ Links ]

WEESE J.S.; DUJIKEREN E. Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine. Veterinary Microbiology, v.140, n.3-4, p.418-429, 2010. Available from: https://www.ncbi.nlm.nih.gov/pubmed/19246166 >. Accessed: Dec. 29, 2016. doi: 10.1016/j.vetmic.2009.01.039. [ Links ]

ZACARCHENCO, P.B et al. “Requeijão” culinário: Historic, market and technological aspects. Revista das Indústrias de Laticínios, v.18, p.70-74, 2012. Available from: http://revistalaticinios.com.br/wp-content/uploads/2011/09/56-76-Fazer-Melhor.pdf >. Accessed: Dec. 23, 2016. [ Links ]

0CR-2017-0008.R2

Received: January 04, 2017; Accepted: March 31, 2017; Revised: April 28, 2017

E-mail: gabrielrossiveterinario@gmail.com. *Corresponding author.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License