numerical taxonomy of microorganisms isolated from goat cheese made in chile

Numerical Taxonomy of Microorganisms Isolated from Goat CheeseMade in Chile

Bernardo Prado,1 Alicia Jara,1 Ana del Moral,2 Eliana Sa´nchez1

1Laboratory of Microbiology, Universidad Te´cnica Federico Santa Marı´a, Sede Vin˜a del Mar, Vina del Mar, Chile2Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain

Received: 20 February 2001 / Accepted: 12 April 2001

Abstract. 118 strains of heterotrophic microorganisms were isolated from goat cheese produceddomestically in the IV Region of Northern Chile (Serene, Ovalle, and Illapel) and sold in supermarketsin Valparaı´so, Chile. The results of 89 phenotypic tests were numerically analyzed against 17 referencestrains, using the simple matching coefficient (SSM). Thirteen phena were found at a 78% similarity level.Five of them (A, B, C, D, and E) were assigned to the familyEnterobacteriaceae, phenon F wasidentified as belonging to the genusAeromonas and strains of phenon G were assigned to the genusAcinetobacter. The other phena were identified as being members of the generaBacillus (H, I, and J),Staphylococcus (K), Enterococcus (L), andMicrococcus (M). Approximately 19% of the isolates wereEscherichia coli and 27%,Staphylococcus aureus.

In Latin America there are more than 19 goat species,most of which are herded in the barren regions. Thesocial importance of this is easy to understand as goatproducts are often the population’s only source of in-come and more than 315,000 families earn their living byselling goat cheese. Nevertheless, a general lack of hy-giene and sanitation prevails at all stages of agro-indus-trial production and processing.

Goat breeding in Coquimbo in Norte Chico (Chile)is fairly unsophisticated and the death rate of these ani-mals from parasites and other infections is quite high. InChile, very little sanitary control is imposed upon pro-duce sold to the public [5, 3, 15]. According to Furtado[6], when milking is not done under sanitary conditionsthe milk may contain more than 2� 106 microorganismsper ml. This count may increase considerably if adequatesanitary conditions are not met during the manufacture,transport, and retail of the cheese. Manufacturing stan-dards and conditions greatly influence the microbial con-tent of goat cheeses [8].

Domestically produced goat cheese, usually madeunder very rudimentary conditions, is widely consumedthroughout Chile. It is made with unpasteurized milk andgenerally sold in cheeses weighing between 600 and

1000 grams. The lack of sanitary standards in this type ofcheese has been the suspected cause of many outbreaksof gastroenteritis [3], especially in the Central Zone ofChile in the spring and summer months.

Information is scarce concerning the total micro-biota contained in goat cheese in Chile and its effects onpublic health [2, 19]. We have carried out a numericalstudy of the taxonomy of such cheeses to determine themain bacterial groups and their potential effects on thehealth of consumers.

Materials and Methods

Samples. A total of 40 samples of goat cheese of 200 g each werebought from supermarkets in Valparaı´so, Chile, during the marketingperiod from October 1997 to April 1998. The samples were transportedto the laboratory under refrigeration, where they were kept at 4°C andanalyzed within 24 h.

Microbiological analysis. The rind was discarded and 25 g samples ofcheese were homogenized with 225 ml of sterile citrate solution (2%wt/vol). Serial dilutions were prepared from these samples in sterile,quarter-strength Ringer solution. We used the pour-plate technique,inoculating 1 ml of the appropriate dilution onto Tripticase soy agar(TSA, Difco). The plates were incubated at 25°C for 72 h [1, 10].

Bacterial strains. A total of 118 strains were isolated from goat cheesebought from supermarkets in Valparaı´so, Chile. These strains includedGram-negative and Gram-positive, aerobic, and facultative anaerobicbacteria. The reference strains used were:Klebsiella pneumoniaeCorrespondence to: Ana del Moral;email: admoral@platon.ugr.es

CURRENT MICROBIOLOGY Vol. 43 (2001), pp. 396–399DOI: 10.1007/s002840010326 Current

MicrobiologyAn International Journal© Springer-Verlag New York Inc. 2001

NCTC 7761, Enterobacter aerogenes NCTC 10006, Escherichia coliATCC 9637, Enterobacter cloacae NCTC 10005, Serratia marcescensNCTC 1377, Salmonella typhi ATCC 19430, Salmonella paratyphiNCTC 8388, Aeromonas sobria CCM 2807, Aeromonas hydrophilaNCMB 86, Proteus mirabilis NCTC 5887, Proteus vulgaris NCTC4635, Bacillus subtilis NCTC 8236, Bacillus cereus CCM 2010, Ba-cillus pumillus NCTC 10337, Staphylococcus aureus NCTC 6571,Enterococcus faecalis ATCC 27285, and Micrococcus luteus NCTC2565.

Characterization of the isolates. 101 phenotypic characteristics weredetermined for each strain using morphological, physiological, bio-chemical, and nutritional tests [24].

Numerical analysis. 89 characteristics were selected for numericalanalysis. Positive and negative results were coded as 1 and 0 respec-tively. Non-comparable and missing data were coded as 9. Strainsimilarities were estimated by the simple matching coefficient (SSM)[20] and clustering was done by the unweighted pair-group method ofassociation (UPGMA) [21].

Results and Discussion

The results obtained for counts on violet-red bili glucosemedium (VRBG) are 2.7 � 106 UFC/gr and on platecount agar medium (PCA) are 6.5�108 UFC/gr.

Using the SSM coefficient and UPGMA clustering,all the isolates were grouped into 13 clusters at a simi-larity level of 78%. Ten of the reference strains weregrouped with the isolates from goat cheese (Fig. 1). Onlyseven reference strains did not cluster at the 78% simi-larity level. The use of non-selective media in our assaysresulted in our being able to isolate a wide range ofmicroorganisms belonging to very different genera fromour goat-cheese samples. Most of the genera belonged tothe family Enterobacteriaceae (phena A, B, C, D, and E)and Micrococacceae (phena K, L, and M) and, to a lesserextent, to the genera Bacillus (phena H, I, and J), Acin-etobacter (phenon G), and Aeromonas (phenon F) (Fig.1). It must be understood that these cheeses are madewith fresh, unpasteurized milk thus, our results tend toagree with those obtained by other authors who havereported the presence of different genera of the familyEnterobacteriaceae in rural goat milk [3, 5, 6, 14, 15,25]. Above all, it is the presence or absence of this familyof bacteria that is used as an indicator of the safe sanitarycondition or otherwise of food.

One of the phena identified was Escherichia coli(phenon D), the presence of which disqualifies food asmarketable by the National Health Service of Chile. E.coli is often found in association with Salmonella and,although we did not isolate Salmonella on this occasion,we did so in a previous study when using selective andenrichment media [16].

The genera Proteus (phenon E), Citrobacter (phe-non B), Enterobacter (phenon C), and Klebsiella (phe-non A) are present in many areas of the environment but

especially in the intestine of both man and animals.Members of these genera may cause gastrointestinal orother infections, usually by transmission through food-stuffs [23].

Phenon F has been identified as Aeromonas hy-drophila because of its phenotypic similarity to the mem-bers of this species. Furthermore, the type strain groupedinto this phenon, Aeromonas hydrophila, isolated fromgoat milk in England, has been cultured at low temper-atures and therefore has been characterized as beingpsychrotrophic [9]. Aeromonas hydrophila isolated inour study grew best at between 2°C and 42°C and there-fore might be considered a psychrotrophic-mesophilicorganism, with a wide temperature range for growth. Thepresence of Aeromonas in goat cheese may be due toenvironmental contamination since it is commonly foundin soil and water.

Fig. 1. Simplified dendrogram showing the clustering of strains into 13phena, based on the SSM coefficient and unweighted average linkageclustering (UPGMA) for 118 microorganisms isolated from goatcheese produced domestically in the IV Region, Chile.

B. Prado et al.: Goat Cheese Microorganisms 397

Phena H, I, and J represent three species of the genusBacillus, B. subtilis, B. cereus, and B. pumillus. Theorganisms of this genus are often abundant and widelydistributed because of their capacity to form spores ca-pable of resisting adverse conditions. Bacillus cereus iswell recognized as being a potential food-poisoningagent. It has been found occasionally in pasteurized milkproducts [11].

We have identified phenon K as Staphylococcusaureus and, with 29 isolates, it was the most widelyrepresented in our analyses. The toxins produced by S.aureus are thermostable and very persistant [22]. Thismicroorganism is frequently found in large quantities inunpasteurized milk, which would account for its notablepresence in our goat cheese [17]. Its presence in foodnormally indicates that the raw material is an importantsource of contamination but it can also derive fromhuman hands, noses, or wounds, as well as from aninfected animal. Nunez et al. [15] and Camacho andSierra [3] also found large numbers of S. aureus inhomemade goat cheese from Chile. Other studies havefound S. aureus to be a cause of food poisoning fromhomemade cow cheese in Costa Rica [4]. The frequentcases of food poisoning from goat cheese could be due tothe ingestion of the toxin produced by S. aureus [13].

Phenon L, comprising six strains, has been classifiedwithin the genus Enterococcus. Enterococcus is widelydistributed throughout many different types of foods,especially those of animal origin and, therefore, the de-termination of E. faecalis and of E. faecium is a tradi-tional indicator of fecal contamination [18]. In our study,the presence of E. faecalis in the goat cheese clearlyindicates a lack of sanitary conditions during the manip-ulation of the cheese.

Phenon M has been included within the genus Mi-crococcus, which is commonly found on the skin of mostmammals and thus may derive either from cheese han-dling or from the goats themselves. In a numerical tax-onomy of isolates from Spanish cheeses, Garcıa et al. [7]have reported the presence of Micrococcus. Species ofthis genus are often considered to be opportunistic patho-gens, especially in immunocompromised patients.

A comparison of our results with those of Litopou-lou-Tzanetakis and Tzanetakis [12], who applied selec-tive media to the same product, reveals the wide micro-bial diversity found in goat cheese in Chile, with thefamily Enterobacteriaceae being highly represented. Inour study we have also detected the presence of generanot encountered by these authors, such as Aeromonas,Bacillus, Staptayhlococcus, and Micrococcus.

This large number of contaminant organisms clearlyindicates the need for much stricter hygiene controlsthroughout the whole chain of manufacture, manipula-

tion, transport, and sale of domestically producedcheeses in Chile.

ACKNOWLEDGMENT

The authors wish to thank A.L. Tate for translating and revising thetext.

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