Revista de Saúde Pública
I SSN 0034- 8910 versão im pressa
Rev Saúde Pública 2003; 37(6)
Microbiological quality of hum an m ilk from a Braz ilian m ilk bank
Álvaro B Serafini, Maria Cláudia D P B André, Márcia A V Rodrigues, André Kipnis, Cynthia O Carvalho, Maria Raquel H Cam pos, Érica C Monteiro, Fábia Martins and Thiago F N Jubé
Departam ento de Microbiologia, I m unologia, Parasitologia e Patologia do I nstituto de Patologia Tropical e Saúde Pública da Universidade Federal de Goiás. Goiânia, GO, Brazil
ABSTRACT
Objective
The obj ect ives of t he present st udy were t o det erm ine t he prevalence of pot ent ially pat hogenic m icroorganism s t hat indicat e t he hygienic and sanit ary condit ions of hum an m ilk sam ples collect ed at a Human Milk Bank.
Methods
Three hundred and thirty eight (338) sam ples of hum an m ilk collected from a m ilk bank in a m aternity in the m unicipality of Goiânia, in the state of Goias, Brazil were subm itted to m icrobiological analysis. The lat t er were plat ed on McConkey agar according t o t he t ype of bact eria. Am ong t he t ot al num ber of sam ples collected, 194 consisted of raw m ilk and the rem aining 144 were pasteurized m ilk.
The presence of St aphylococcus spp., St rept ococcus spp., yeast s and m olds, and Ent erobact eriaceae w as v erified in the raw m ilk sam ples. Staphylococcus aureus were isolated in 10 ( 5.2% ) sam ples, St aphylococcus epiderm idis in 28 ( 14.4% ) sam ples, St rept ococcus spp. in t hree ( 1.6% ) sam ples, yeasts and m olds in 43 (22.2% ) and Enterobacteriaceae in 49 (25.3% ) sam ples. I n a hundred and fort y four ( 144) sam ples which underwent t herm al t reat m ent St aphylococcus aureus was det ect ed in five ( 3.5% ) sam ples, St aphylococcus epiderm idis in 15 ( 10.4% ) , St aphylococcus lugdenensis in t wo ( 1.4% ) , St rept ococcus spp. in four ( 2.8% ) , yeast s and m olds in 37 ( 25.7% ) , and Ent erobact eriaceae in nine (6.3% ).
Conclusions
Analysis indicated a high degree of contam ination in raw hum an m ilk, and as for the pasteurized m ilk, despit e elim inat ion of t he great m aj orit y of pot ent ially pat hogenic m icroorganism s, the percentage of yeasts and m olds was higher than in raw m ilk, dem onstrating that a lower degree of initial cont am inat ion would be necessary for past eurizat ion t o be an efficient m eans of m icrobiological cont rol.
Keywords
Milk banks. Milk , hum an. Quality control.
I N TRODUCTION
Exogenous infections, originating from the hospital environm ent, deserve greater attention from public healt h professionals t han endogenous ones, for hum a n beings are t he sources of t he agent s t hat provoke t hem . The form er include hospit al em ployees, pat ient s and visit ors as well as hospit al instrum ents and equipm ent.5 Since it is very difficult t o im plem ent m easures t o cont rol hospit al infect ions in all sect ors of t he hospit al sim ult aneously, effort s should be direct ed prim arily t owards higher risk sect ors, such as nurseries, nut rit ional services, surgical wards, int ensive care unit s and cent ers of st erilizat ion.
The m icrobiological quality of expressed m ilk distributed by these m ilk banks is a public health issue, for the children who will consum e t his product have low resist ance t o neonat al infect ions. 1 , 2 , 1 3 , 2 1 The
m ost im portant issue concerning HMBs is the bacteriological control of donated m ilk,1 1 for
consum ption of contam inated hum an m ilk m ay be the cause of neonatal diseases.2 3
I t is im port ant to obtain m ore epidem iological data on bacterial contam ination of hum an m ilk and the developm ent of educat ional work wit h t he m ot hers, nurses, nurses’ aides, nut rit ionist s, pediat ricians, and int ensive care physicians, m ust be carried out so as t o m ake them aware of the risks involved in the preparation and consum ption of hum an m ilk.2 1 The purpose of t his st udy is t o invest igat e t he
prevalence of m icroorganism s in m ilk being offered by a hum an m ilk bank so as to subsidize the com pet ent aut horit ies conduc t ing program s for t he prevent ion of neonat e infect ions.
M ETHODS
Three hundred and thirty eight (338) sam ples of hum an m ilk were collected from a m ilk bank in a m aternity in the m unicipality of Goiânia, in the state of Goias. Am ong the total num ber of sam ples collected, 194 consisted of raw m ilk and the rem aining 144 were pasteurized m ilk. These sam ples were packaged and sent im m ediately to the Laboratório de Microbiologia de Alim entos do Depart am ent o de Microbiologia do I nst it ut o de Pat ologia Tropical e Sa úde Pública de Goiânia ( Laborat ory of Food Microbiology of t he Depart m ent of Microbiology of Tropical Pat hology and Public Health of Goiania) , where they were subm itted to m icrobiological analysis.
Due t o t he quant it y of sam ples and t he fact t hat t he m at erial in quest ion was produced by secret ion, procedures adopted for m icrobiological analysis were those indicated by Konem an et al1 4 ( 1997) with sam ples being plat ed init ially on blood agar and McConkey’s agar and, according t o t he t ype of bact eria isolat ed, posterior identification was undergone in appropriate m edium s. Bacteria pertaining t o t he St apph y lococci gender were t est ed for t heir capacit y t o produce coagulase and resist ance t o
novobiocin. As t o t he St r ep t ococci, based on hem olysis, t est s of resist ance t o bacit racin, opt ochin and
ot her biochem ical t est s were conduct ed. I n order t o ident ify gram- negat ives, screening was undertaken in triple sugar iron ( TSI ) agar and identification was obtained by m eans of biochem ical t est s. Molds and yeast were ident ified by m eans of a t echnique indicat ed by t he Am erican Public Healt h Associat ion2 5, which consist ed in plat ing t he sam ples on a 10% pot at o dext rose agar, acidified by t art aric acid unt il t he pH was 3.5, and incubat ing it at 25o C+ 1 for 5 t o 7 days.
RESU LTS
Potentially pathogenic and/ or com m ensal organism s were identified in 70.4% ( 136 / 194) of the raw m ilk sam ples and 50.7% (73/ 144) of the sam ples of pasteurized m ilk (see Table 1).
Table 1 - Percentage of positive and negative samples found in raw and pasteurized expressed human milk (EHM) from a milk bank in Goiania, GO.
Microorganisms Not pasteurized EHM Pasteurized EHM
N % N %
Positive 136 70.1% 73 50.7%
Negative 58 29.9% 71 49.3%
Total 194 100% 144 100%
Am ong the sam p les of raw m ilk, St r ep t o co cci of the viridans group were isolated in three ( 2.2% ) ; 10
sam ples ( 7.35% ) presented colonies of St aphy lococcus aur eus; S. epiderm idis were isolated in 28
(20.59% ) sam ples; and 49 (36.0% ) sam ples were contam inated by Ent er obact er iaceae. Furtherm ore,
43 ( 31.6% ) sam ples of raw m ilk were contam inated by m olds and yeast ( see Table 2) .
Table 2 - Distribution of the results of microbiological analysis of the samples of expressed raw and pasteurized human milk, collected in milk banks from 1999 to 2001, in Goiania, GO.
Microorganismo Not pasteurized EHM Pasteurized EHM analyzed positives analyzed positives
N % % N % %
Staphylococcus aureus 10 5.2 7.4 5 3.5 6.9
S. epidermidis 28 14.4 20.6 15 10.4 20.6
S. lugdenensis - - - 2 1,4 2,7
Streptococcus viridans group 3 1.6 2.2 2 1.4 2.7
S. pyogenes - - - 2 1.4 2.7
Klebsiella sp 3 1.6 2.2 1 0.7 1.4
K. oxit oca 3 1.6 2.2 1 0.7 1.4
K. rhinoscleromatis 6 3.1 4.4 - - -
K. pneumoniae 4 2.1 2.9 2 1.4 2.7
Enterobacter sp 1 0.5 0.7 - - -
E. aer ogenes 2 1.0 1.5 - - -
E. agglomerans 3 1.6 2.2 - - -
E. cloacae 12 6.2 8.8 3 2.1 4.1
E. hormaechei 2 1.0 1.5 - - -
Escherichia coli 3 1.6 2.2 - - -
Citrobacter freundii 1 0.5 0.7 - - -
C. diversus 1 0.5 0.7 - - -
Morganella morganii 1 0.5 0.7 - - -
Serratia marcescens 3 1.6 2.2 - - -
S. liquefaciens - - - 1 0.7 1.4
Proteus mirabilis 1 0.5 0.7 1 0.7 1.4
Hafnia alvei 2 1.0 1.5 - - -
Pantoea sp. 1 0.5 0.7 - - -
Pseudomonas sp. 3 1.6 2.2 1 0.7 1.4
Molds and yeast 43 22.2 31.6 37 25.7 50.7 Total: analyzed samples 194 100 - 144 100 - Total: positive samples 136 70.4 - 73 50.7 -
Microorganism s were identified in seventy - three of the 144 sam ples of pasteurized m ilk from the HMB. Am ong these, St aphy lococcus lugdenensis, St r ept ococcus py ogenes, and St r ep t ococcu s of t he vir idans group were isolated in two (2.7% ) sam ples; St aphy lococcus aur eus in five (6.9% ) sam ples; S. epiderm idis in 15 (20.6% ) and En t er obact er iaceae were det ect ed in 12.3% of t he sam ples.
Furtherm ore, 37 (50.7% ) sam ples tested posit ive for m olds and yeast s ( see Table 2) .
The presence of high levels of contam inants in raw hum an m ilk entails in a reduction of its biological benefits because its nutrients are being utilized by the m icroflora present in the m ilk, thus diminishing its protective im m unological qualities.2 These sit uat ions, in t he m aj orit y of cases, lead t o t he
classificat ion of t his product as im proper for consum pt ion, t aking int o account t he vulnerabilit y of t he recipient infant s. Furt herm ore, t he great er the quantity and diversity of m icroorganism s present in the m ilk, t he less efficient will t he out com e of t he process of past eurizat ion be. I t m ay be not ed t hat t here is a great gap in t he crit eria for select ion of m ilk for past eurizat ion.3
The prevalence of St aphy lococcus aur eus in the oropharynx of hum an beings varies from 35to 40%
and in the m outh and saliva its prevalence varies from 10 to 35% . However, it’s presence in hum an m ilk m ay be int erpret ed as being due t o cont am inat ion from a secondary source, such as t he skin and nasal fossa, or unsat isfact ory hygienic or sanit ary condit ions of t he ut ensils em ployed in it s m anipulat ion. The great est concern wit h respect t o it s presence is t he occurrence of st rains which produce t oxins t hat are resist ant t o past eurization.3
Pereira et al ( 1995) report ed t he presence of St aph y lococcu s in al the sam ples of m aternal m ilk
collected from 19 wom en with sym ptom s of m astitis. Am ong the 19 sam ples, 8 synthesized detectable quant it ies of ent erot oxins, being t hat in som e cases, it was observed t hat t hey also produced t he t oxin of t he t oxic chock syndrom e.
As t o t he raw m ilk, t he result s present ed in t his st udy are sim ilar t o t hose report ed by Caroll et al7
( 1979) who observed S. aureus in 13 (6.2% ) of the 207 sam ples of expressed hum an m ilk. Other
researchers found coagulase posit ive St aph y lococcu s in different proportions of hum an m ilk sam ples
subm itted to analysis: 28.1% (Nikodem uz1 6), 29% (Alm eida et al3), 51.7% (Alm eida et al2) and
furtherm ore, am ong 7,570 sam ples, bacterial growth was observed in 230, being that 40% of these were S. aur eus.8
Wy at t and Mat a2 6 ( 1969) , isolat ed ent erobact erias in 18% of t he 51 sam ples of colost rum t hey
subm it t ed t o analysis, revealing t he low level of personal hygiene and sanit ary condit ions of t he st udy population, living in the sm all village of Santa Maria Cauqui in Guatem ala.
Ot her aut hors found E. coli in 8.5% of the 59 sam ples of m aternal m ilk2 2, in 2% of 44 sam ples9,
indicat ing faecal cont am inat ion of t he m ilk, and ent erobact erias in 15 ( 7% ) sam ples. The present result s reveal t he presence of ent erobact erias in 25.3% of t he sam ples of raw m ilk analyzed and in 6.2% of the sam ples of pasteurized m ilk. Therefore, they presented inferior hygienic conditions than t he st udies cit ed above.
Amo ng t he ent erobact erias, coliform s have been singled out as part icularly im port ant in bact eriological cont rol of HMBs, since t heir presence m ay indicat e faecal cont am inat ion, even if it originates from an indirect source and doesn’t necessarily im ply in the isolat ion of E. coli.2 Research
has indicated that contam ination by coliform m icroorganism s m ay originate from the environm ent, as observed in a st udy in which t his group of bact eria were isolat ed in 38.4% of a t ot al of 472 sam ples1 7, and am ong a t ot al 5,710 sam ples of hum an m ilk, coliform s were detected in 1,139 (19.9% )1 7 and in a
total of 837 sam ples, 71 ( 8.48% ) were contam inated with coliform m icroorganism s, whereas only t hree of t hese were E. coli.1 8 I n t his st udy, coliform s were present in 21.1% of t he sam ples of raw
m ilk and in 5.6% of the pasteurized m ilk sam ples, indicating that the efficiency of pasteurization m ay be com prom ised by an initially high count of m icrobes.
The presence of m olds and yeast in foods m ay also indicat e cont am inat ion originat ing from the environm ent or resulting from handling in inadequate hygienic and sanitary conditions.1 5 Almeida4
t hat 230 ( 6.5% ) of a t ot al of 7,570 sam ples analyzed cont ained m olds and yeast .8 The result s of t his
st udy, in which m olds and yeast were det ect ed in 22% of t he sam ples of raw m ilk, are inferior t o t hose observed by ot her aut hors. However, t he percent ages of m olds and yeast det ect ed in t he pasteurized m ilk ( 25.7% ) are a m atter of concern, for they indicate a possible environm ental cont am inat ion occurring aft er t he process of past eurizat ion or t hat t his process m ay be ineffect ive in it self. The presence of pat hogenic yeast in past eurized hum an expressed m ilk suggest s t hat t his could be a source of infect ion t o neonat es during breast feeding. Qualit y cont rol by m eans of yeast count s in expressed hum an m ilk m ay be a good indicat or of problem s relat ed t o hygiene, st orage or t ransport at ion.2 1
I nadequat e past eurizat ion not only presum ably endangers t he beneficial propert ies of expressed hum an m ilk, but m ay also increase the susceptibility to subsequent contam ination.6
The RDC ( Resolução da Diret oria Colet iva) [ Resolut ion of t he Collect ive Direct ory] Resolut ion no 12 ,
inst it ut ed by t he Minist ry of Healt h1 4 in January, 2001, establishes criteria for the m icrobiological
control of hum an m ilk for the first tim e in Brazil. According t o t his resolut ion, t he quant it y of viable m esophilic aerobic m icroorganism s perm itted is up to 100 CFU/ m L, coliform s at 35oC and St aphy lococcus coagulase- positive absent in 1 m L and Salm onella sp absent in 25 m L. According t o
t his r esolut ion, 4.9% of the sam ples analyzed in the present investigation were im proper for hum an consum pt ion, for t hey did not present t he analyt ical param et ers est ablished in Annex 1 of t he aforem ent ioned Resolut ion. Taking int o considerat ion t he result s obt ained, t he aut hors believe t hat efforts should be m ade to im prove the m icrobiological control of expressed hum an m ilk, including the m ilk which is going to be pasteurized. I n this sense, m ore rigorous m easures for m onitoring the quality of hum an m ilk are indispensable so as t o guarant ee safe feeding for neonat es.
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Address to correspondence
Álvaro Bisol Serafini
I nst it ut o de Pat ologia Tropical e Saúde Pública Rua Delenda Rezende de Melo, s/ n Setor Universitário 74605 - 050 Goiânia, GO, Brazil
E- mail: abisol@ipt sp.ufg.br
Financiado pela Fundação de Apoio à Pesquisa/ FUNAPE/ UFG ( Processo n 66.357) . Received on 23/ 1/ 2002. Reviewed on 27/ 6/ 2003. Approved on 3/ 7/ 2003.
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