Almeida, G.

Hogg, T.

Leite, D.

Magalhães, R.

Silva, J.

Teixeira, P.

English

Repositório Institucional da Universidade Católica Portuguesa

Characterization of L. monocytogenes isolatedfrom traditional Portuguese cheesesR. Magalhães, G. Almeida, D. Leite , J. Silva, T. Hogg and P. TeixeiraUniversidade Católica Portuguesa · Escola Superior de BiotecnologiaPaula TeixeiraRua Dr. António Bernardino de Almeida  · 4200-072 PORTOTel.: + 351 225 580 00 45  · Fax: + 351 225 580 111E-mail: pcteixeira@esb.ucp.ptIntroductionL. monocytogenes is considered a serious public health hazard. The incidence of human listeriosis is low (2-15 per million population) but the death rate among individuals at risk approaches 30%, far exceeding that of other common food-borne pathogens. Consumption of contaminated foods is probably the primary source of human listeriosis and the prevalence of L. monocytogenes in foods is relatively high. The aim of this study was the characterisation of 62 strains of L. monocytogenesisolated from 12 different Portuguese commercial cheeses according to the serotype, biotype, RAPD-PCR and resistance to antibiotics. Materials and MethodsResults and DiscussionTable 1 shows the Minimal inhibitory concentration of various antibiotics(MIC50 and MIC90). 90% of the isolates were found with a MIC of 128 μg/ml for nitrofurantoin. With the exception of penicillin and ampicillin, there are no breakpoints defined for antibiotics resistance L. monocytogenes. Therefore it was not possible to classify them according to their susceptibility to the antibiotics. However it was possible to conclude that there are differences between the strains and also between the origin of the strains and the susceptibility to antibiotics.Table 2 shows the biotype and the serogroup determined for each L. monocytogenes strain. In general, the isolates from each cheese  demonstrated a similar pattern.For all the analysed cheeses and concerning the sensitivity to heavy metals three biotypes were present: As:s,Cd:s (90,5%); As:s,Cd:r (1,6%) and As:r,Cd:s (7,9%). Four serovars were found: 1/2a-3a (4,8 %), 1/2b-3b (71,4%), 1/2c-3c (1,59%) and 4b-4d-4e (21,9%).The isolates were analysed and characterised according to the RAPD-PCR profile using primer UBC155. 5 different clusters were observed. Isolates from different cheeses were often included in  the same cluster .ConclusionsThis study demonstrated that other typing methods are necessary to be used in order to identify possible correlations between isolates.References(1) Anonymous, 1996. VIDAS Listeria monocytogenes. AFNOR, Bio 12/3-03/96.(2) Anonymous, 1996. Microbiology of food and animal feeding stuffs - horizontal method for detection and enumeration of Listeria monocytogenes: Part 1. Detection Method. International Standard ISO 11290-1. International Organization for Standardization, Geneva.(3) Vaz-Velho, M., Duarte, G., McLauchlin, J. and Gibbs P., 2001. J. Appl.  Microbiol. 91:556-562. (4) McLauchlin, J., Hampton, M. D., Shah, S.,Threlfall, E. J.,Wieneke, A. A. and Curtis, G. D.W., 1997. J. Appl. Bacteriol. 83:381-388.(5)Doumith M., Buchrieser C., Glaser P., Jacquet C. and Martin P., 2004. J. Clin. Microbiol. 42 : 3819-3822AcknowledgmentsThis work was funded by Fundação para a Ciência e Tecnologia project POCTI/AGR/44889/2002Table 2- Characterization of cheese isolated strains.isolate cheese Serogroup As Cd Tetra1 Q1 1/2a-3a - - -2 1/2b-3b - - -3 1/2b-3b - - - 4 1/2b-3b - - - 5 1/2b-3b - - - 6 1/2b-3b - - - 7 Q2 1/2b-3b - - - 8 1/2b-3b - - - 9 1/2b-3b - - - 10 1/2b-3b - - - 11 1/2b-3b - - - 12 1/2b-3b - - -13 4b-4d-4e - - - 14 4b-4d-4e - - -15 4b-4d-4e - - - 16 Q3 4b-4d-4e - - - 17 4b-4d-4e - - - 18 4b-4d-4e - - - 19 4b-4d-4e - - - 20 4b-4d-4e - - - 21 1/2b-3b - - - 22 Q4 1/2b-3b - - - 23 1/2b-3b - - - 24 Q5 1/2b-3b - - - 25 1/2b-3b - - - 26 1/2b-3b - - - 27 Q6 1/2b-3b - - - 28 1/2b-3b - - -29 1/2b-3b - - - 30 1/2c-3c - - - 31 1/2b-3b - - - 32 1/2b-3b - - + 33 1/2b-3b - - -34 1/2b-3b - - - 35 1/2b-3b - - - 36 Q7 1/2b-3b - - - 37 1/2b-3b - - - 38 1/2b-3b - - - 39 1/2b-3b - - - 40 1/2b-3b - - -41 1/2b-3b - - -42 Q8 1/2a-3a - - - 43 1/2a-3a - - - 44 1/2b-3b - - - 45 Q9 1/2b-3b - - - 46 1/2b-3b - - -47 1/2b-3b - - - 48 1/2b-3b - - - 49 1/2b-3b - - -50 1/2b-3b - - - 51 1/2b-3b - - -52 Q10 1/2b-3b - - - 53 1/2b-3b - - - 54 1/2b-3b - - - 55 1/2b-3b - - - 56 1/2b-3b - - - 57 1/2b-3b - - -58 Q11 4b-4d-4e - - -59 4b-4d-4e + - -60 4b-4d-4e + - -61 Q12 4b-4d-4e + - -62 4b-4d-4e + - -Resistance to As, Cd and Tetra Figure 1- Dendrogram obtained by RAPD-PCR. Table 1- MIC50 and MIC 90 for strains isolated from commercial cheeses MIC (µg/ml) for strain isolated from commercial cheeses (n=62) Drug Range 50% 90% Penicillin G 0,25 – 2 0,25 1 Ampicillin 0,0625 – 1 0,25 0,5 Gentamicin 0,03125 – 1 0,125 0,125 Ciprofloxacin 1 – 128 1 2 Erytromycin 0,5 – 16 1 1 Vancomycin 0,5 – 1 1 1 Choramphenicol 2 – 8 4 8 Tetracycline 1 – 128 4 4 Rifampicin 0,03125 – 0,25 0,125 0,25 Nitrofurantoin 32 – 128 128 128  L. monocytogenes detection by:Subtyping of L. monocytogenes isolates Serogroupidentification by Multiplex PCR (Doumith et al, 2004)Resistance  to arsenic, cadmium and tetracycline (Vaz-Velho et al. 2001)MPN technique Direct enumeration Vidas (biomérieux)Resistance to antibiotics(NCCLS, 2005)

Characterization of L. monocytogenes isolated from traditional portuguese cheeses

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Characterization of L. monocytogenes isolated from traditional portuguese cheeses

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