Pulsed-Field Gel Electrophoresis characterization of Listeria monocytogenes isolates from cheese manufacturing plants in São Paulo, Brazil

doi:10.1016/j.ijfoodmicro.2013.12.018

International Journal of Food Microbiology

Volume 173, 3 March 2014, Pages 21-29

https://doi.org/10.1016/j.ijfoodmicro.2013.12.018 Get rights and content

Highlights

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L. monocytogenes isolates from 3 cheese manufacturing plants in Brazil were evaluated.
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Isolates were classified in 4 serotypes, with predominance of serotype 4b.
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PFGE analysis showed different sources of contamination and persistence in 2 plants.
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L. monocytogenes was widely distributed and adapted to the dairy plant environment.

Abstract

This study aimed to evaluate the occurrence of Listeria monocytogenes in cheese and in the environment of three small-scale dairy plants (A, B, C) located in the Northern region state of São Paulo, Brazil, and to characterize the isolates using conventional serotyping and PFGE. A total of 393 samples were collected and analyzed from October 2008 to September 2009. From these, 136 came from dairy plant A, where only L. seeligeri was isolated. In dairy plant B, 136 samples were analyzed, and L. innocua, L. seeligeri and L. welshimeri were isolated together with L. monocytogenes. In dairy plant C, 121 samples were analyzed, and L. monocytogenes and L. innocua were isolated. Cheese from dairy plants B and C were contaminated with Listeria spp, with L. innocua being found in Minas frescal cheese from both dairy plants, and L. innocua and L. monocytogenes in Prato cheese from dairy plant C. A total of 85 L. monocytogenes isolates were classified in 3 serotypes: 1/2b, 1/2c, and 4b, with predominance of serotype 4b in both dairy plants. The 85 isolates found in the dairy plants were characterized by genomic macrorestriction using ApaI and AscI with Pulsed Field Gel Electrophoresis (PFGE). Macrorestriction yielded 30 different pulsotypes. The presence of indistinguishable profiles repeatedly isolated during a 12-month period indicated the persistence of L. monocytogenes in dairy plants B and C, which were more than 100 km away from each other. Brine used in dairy plant C contained more than one L. monocytogenes lineage. The routes of contamination were identified in plants B and C, and highlighted the importance of using molecular techniques and serotyping to track L. monocytogenes sources of contamination, distribution, and routes of contamination in dairy plants, and to develop improved control strategies for L. monocytogenes in dairy plants and dairy products.

Introduction

Listeria monocytogenes is an important human pathogen that occurs in several food processing environments, including dairy processing plants (Kathariou, 2002). Listeriosis has been recognized as a serious public health hazard, with high mortality rates in susceptible individuals, such as the elderly and immunocompromised (Swaminathan and Gerner-Smidt, 2007). Consumption of contaminated dairy products has been associated with cases and outbreaks of human listeriosis (Leite et al. 2006). In Brazil, human listeriosis is underdiagnosed and underreported (Silva et al., 2010), and there is no report on foodborne cases (Brito et al., 2008), although L. monocytogenes is frequently isolated from dairy products (Zaffari et al., 2007, Brito et al., 2008, Abrahão et al., 2008, De Nes et al., 2010, Barancelli et al., 2011).

The main types of Brazilian commercial cheeses include Minas Frescal (non-ripened, fresh cheese) and Prato (ripened cheese) varieties, accounting for nearly 30% of the 640,000 kg of cheese produced annually in the country. However, only Minas Frescal cheese was positive for L. monocytogenes in previous reports (Carvalho et al., 2007, Brito et al., 2008).

The ability of L. monocytogenes to survive long periods in adverse conditions, and to colonize and persist in food processing environments is a threat to the food industry, especially the dairy industry (Miettinen et al., 1999, Kathariou, 2002, Wagner et al., 2006). Pulsed Field Gel Electrophoresis (PFGE) has been successfully used in L. monocytogenes typing in epidemiological surveys (Miettinen et al., 1999, Barret et al., 2006, Neves et al., 2008, Latorre et al., 2009, Sauders et al., 2009), and the association with serotyping is widely recommended in epidemiological studies to assess the persistence of this pathogen in the food processing environment (Chasseignaux et al., 2001).

In Brazil, there is little information about genotypic variation in L. monocytogenes isolated from the environment of dairy industries, especially in small-scale cheese processing plants that produce Minas Frescal and Prato cheeses. Furthermore, there are no studies in Brazil comparing the genetic profile of L. monocytogenes isolated from different dairy plants. In this context, this study aimed to isolate and characterize L. monocytogenes from cheeses and from the environment of three small-scale cheese processing plants located in the Northeastern region of São Paulo State, Brazil, using serotyping and PFGE.

Section snippets

Sampling procedure

The three dairy plants, located in the northeastern region of the state of São Paulo, were not connected, and were located 100 km away from each other. They did not have raw milk suppliers in common. The volume of cow's milk processed per day and the number of farms that supplied milk to the dairy plants were: 7,000 liters and 60 farms (dairy plant A); 12,000 liters and 60 farms (dairy plant B); and 3,000 liters and 55 farms (dairy plant C). Cheese processing was similar in the three plants

Occurrence of Listeria species

The number of samples contaminated with Listeria spp. species in each sampling site is presented in Table 1. None of the samples of water, raw milk, pasteurized milk or curd used in the manufacture of Minas Frescal cheese were positive for Listeria spp. Among samples that tested positive in any of the dairy factories, the percentage of contamination for L. monocytogenes was 7.1% (28/393). In dairy plant A, only the species L. seeligeri was isolated in 4 of 136 samples (2.9%), and it came from

Discussion

Dairy plants B and C, in which cleaning conditions were inferior to those in dairy plant A, showed the greatest incidence of Listeria spp. L. monocytogenes was isolated in all samplings from these dairy plants, whereas the only species isolated in dairy plant A was L. seeligeri. Isolation of Listeria spp. is a relevant finding, as L. monocytogenes may be found in the same sites where other Listeria species are isolated (Kells and Gilmour, 2004).

In Brazil, previous studies showed that the

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support.

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Pulsed-Field Gel Electrophoresis characterization of Listeria monocytogenes isolates from cheese manufacturing plants in São Paulo, Brazil

Highlights

Abstract

Introduction

Section snippets

Sampling procedure

Occurrence of Listeria species

Discussion

Acknowledgements

Food Control

Syst. Appl. Microbiol.

J. Food Prot.

Food Control

Int. J. Food Microbiol.

J. Food Prot.

Food Control

J. Food Prot.

Int. J. Food Microbiol.

J. Dairy Sci.

J. Food Prot.

Int. J. Food Microbiol.

J. Dairy Sci.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Syst. Appl. Microbiol.

Food Microbiol.

J. Food Prot.

Int. J. Food Microbiol.

Microbes Infect.

J. Food Prot.

Occurrence of Listeria monocytogenes in cheese and ice cream produced in the State of Paraná, Brazil

Rev. Bras. de Ciências Farm.