The aim of the present study is to evaluate the importance of sporulated sulphite-reducing anaerobes on mineral water for bottling and thermal use, according the laboratory results obtained during nine years.N/

Coelho, Carla

Ferreira, Daniela

Maciel, Maria Sameiro

Pizarro, Cristina

Ribeiro, Maria João

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

Sporulated sulphite-reducing anaerobes in natural mineral water quality assessment

Repositório Científico do Instituto Nacional de Saúde

SPORULATED SULPHITE-REDUCING ANAEROBES  IN NATURAL MINERAL WATER QUALITY ASSESSMENT Daniela Ferreira, Maria João Ribeiro, Maria Sameiro Maciel, Carla Coelho and Cristina Pizarro Laboratório de Microbiologia, Departamento de Saúde Ambiental, Instituto Nacional de Saúde Doutor Ricardo Jorge, Rua Alexandre Herculano 321, 4000-055 Porto, Portugal  Natural mineral waters have exceptional characteristics and known since ancient times to benefit human health in several disorders (Lourenço, 2012). According to Portuguese legislation (Decree-Law 156/98), these waters are defined as bacteriologically safe, with stable physico-chemical characteris-tics at the source, within the natural variation range, and which differentiate from usual drinking water given their possible beneficial health effects. Currently the use of Portu-guese natural mineral water for bottling and thermal treatments is widespread. Therefore, bacteriological control is mandatory by legislation (Decree-Law 156/98; Ordinance 1220/2000) and done regularly in order to guarantee water quality. All waters covered by these legislations must be bacteriological wholesome, defined by indicator organisms. The quality requirements include the enumeration of sporulated sulphite-reducing anaerobes, which should be absent from these types of waters. The aim of the present study is to evaluate the importance of sporulated sulphite-reducing anaerobes on mineral water for bottling and thermal use, according to the laboratory results obtained during nine years. 13.  The water samples analyzed have a low contamination occurrence, no more than 5% when accounting for all indicators, excluding heterotrophic bacteria.  The level of mineral water contamination was variable over the years. Besides heterotrophic bacteria, P. aeruginosa largely contributed to the sample non-conformity (except in spring water), SRA spores emerged in second or third positions. Overall, less than 1% of total non-conform water samples correspond to the presence of sporulated sulphite-reducing anaerobes. Most mineral water samples contaminated by SRA spores were absent from the other indicator microorganisms. The frequency of SRA spores in our water sampling was low. Given the extent of national use of these waters, SRA spore assessment may still be useful to accomplish quality assurance because they are a long-lived indicator. Decree-Law 156/98. Diário da República DR 131/1998 - Série I-A, de 06/06/1998. Lourenço, C. (2012) Classificação das águas minerais naturais e de nascente engarrafadas na região centro. In: Águas Minerais Naturais e de Nascente da Região Centro. Mare Liberum-Editora, p. 51-70. Ordinance 1220/2000. Diário da República DR 299/2000 - Série I-B, de 29/12/2000. Introduction   Statistical study conducted from a total 5643 water samples for bot-tling (natural mineral water: 1154; spring water: 2475) and thermal use (for mucosae contact: 1087; baths: 927), located in the Galiza-Trás-os-Montes and Centro-Iberian geotectonic zones, analyzed dur-ing the years 2007 to 2015.  Bacteriological quality indica-tors: sulphite-reducing anaerobe (SRA) spores, heterotrophic plate counts (HPC) at 37 and 22°C, coli-form bacteria, Escherichia coli, en-terococci and Pseudomonas aeruginosa. Methodology Results Natural Mineral Water Figure 1. Percentage of total sample conformity (pie I) and frequency of the bacteriological indicators (pies II and III) detected in each type of water. Legend applies to all charts. SRA: sulphite-reducing anaerobes; HPC: heterotrophic plate counts.  Spring Water Thermal water: for mucosae contact Thermal water: for baths  0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria)Coliform bacteriaE. coliEnterococciP. aeruginosaSRA sporesNatural Mineral Water 0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria)Coliform bacteriaE. coliEnterococciP. aeruginosaSRA spores0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%8,02007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria)Coliform bacteriaE. coliEnterococciP. aeruginosa0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria)Coliform bacteriaE. coliEnterococciP. aeruginosaSRA sporesSpring Water Thermal water: for mucosae contact Thermal water: for baths Figure 2. Percentage of total non-conform samples (heterotrophic bacteria was omitted from charts) between 2007 and 2015, together with the parcel corresponding to each bacteriological indicator. Legend applies to all charts. SRA: sulphite-reducing anaerobes. In this mineral water study, the data from spring water for bot-tling was analyzed separately. Conclusions  Natural Mineral Water Spring Water TW for mucosae contact TW for baths Total waters SRA spores only 2 15 9 5 31 SRA spores with other indicators 0 10 0 1 11 Other indicators without SRA spores 327 228 89 38 682 TOTAL 329 253 98 44 724 Table 1. Number of non-conform samples with or without SRA spores. 0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%8,0%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria) Coliform bacteriaE. coli EnterococciP. aeruginosa SRA spores0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%8,0%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria) Coliform bacteriaE. coli EnterococciP. aeruginosa SRA spores0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%8,0%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria) Coliform bacteriaE. coli EnterococciP. aeruginosa SRA sporesReferences 71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa89,8% 10,2%Conform Non-conform18,8%41,1%26,9%1,3%3,6%8,1%0,3%40,1%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa91,0% 9,0%Conform Non-conform24,8%53,4%4,5%1,5%0,8%7,5% 7,5%21,8%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa95,3% 4,7%Conform Non-conform28,1%26,3%1,8%1,8%3,5%10,5%28,1%45,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosaSRA: sulphite-reducing anaerobes; TW: thermal water. 0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform(pathogenic bacteria)Coliform bacteriaE. coliEnterococciP. aeruginosaSRA spores0,0%1,0%2,0%3,0%4,0%5,0%6,0%7,0%8,0%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform (pathogenic bacteria) Coliform bacteriaE. coli EnterococciP. aeruginosa SRA spores0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015% non-conform(pathogenic bacteria)Coliform bacteriaE. coliEnteroc cci. SRA spores71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa71,5% 28,5%Conform Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa89,8% 10,2%Conform Non-conform3,27%7,17%4,69%0,23%0,62%1,41%0,06%7,00%HPC at 37 CHPC at 22 CColiform bacteriaE. coliEnterococciOther indicators than HPCP. aeruginosa71,5% 28,5%Conf rm Non-conform34,5%61,8%1,1%0,2%0,2%0,5%1,6%3,6%HPC at 37°CHPC at 22°CColiform bacteriaE. coliEnterococciSRA sporesP. aeruginosa0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years0,0%,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2 08 2 09 20 2012 2013 2014 2015Non-conform samples (%)Years0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years0,0%0,51,1,52,2,53,03,54,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years0,0%0,5%1,0%1,5%2,02,53,3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years0,0%0,5%1,0%1,5%2,0%2,53,3,544,5%2007 2008 2009 2010 2011 2012 2013 2014 2015Non-conform samples (%)Years0,0%0,5%1,0%1,5%2,0%2,5%3,0%3,5%4,0%4,5%2007 2008 2009 2010 2011 2012 2013 2014 2015total non-conform indicatorsColiform bacteriaE. coliEnterococciP. aeruginosaSRA spores

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Sporulated sulphite-reducing anaerobes in natural mineral water quality assessment

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