BREASTFEEDING MEDICINE Volume 9, Number 10, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/bfm.2014.0046
Breastfeeding Medicine 2014.9:557-558. Downloaded from online.liebertpub.com by ETH Zurich on 01/07/15. For personal use only.
Detection, Antibiotic Resistance, and Pathogenicity of Staphylococci in Samples from a Brazilian Human Milk Bank Jessica Bomfim de Almeida,1,2 Suzi Pacheco de Carvalho,1 Andre´ Luı´s Vivas de Almeida,2 Guilherme Barreto Campos,3 Ma´rcio Vasconcelos Oliveira,2 Jorge Timenetsky,3 and Lucas Miranda Marques1,2
Dear Editor:
S
tudies on Staphylococcus aureus in samples of stored milk used to feed newborns are scarce in the literature.1 Therefore the aim of this study is to detect S. aureus in samples of raw milk, pasteurized milk, and glass bottles collected in a Brazilian human milk bank. The Human Milk Bank of Esau´ Matos Municipal Hospital is located in Vito´ria da Conquista, Bahia, Brazil. It was opened in 2004, when 22.5 L of milk was collected for newborns of the neonatal intensive care unit. Now the hospital has 682 donors providing about 878 L of milk per year. It is considered one of the largest milk banks in the region. All donated milk undergoes a pasteurization process in the service itself. The milking is done manually, and processing includes storing raw human milk in refrigerators in glass bottles previously sterilized by autoclaving, defrosting and rebottling, labeling of bottles, physicochemical quality control, pasteurization at 62.5C for 30 minutes, determining the microbiological quality of pasteurized human milk, and storage, distribution, and portioning. For the analysis, in total, 160 samples were collected. Of these, 80 samples were from raw human milk, 30 from pasteurized human milk, and 50 swabs from glass bottles for storing milk. Physicochemical quality control was performed on the samples. The samples were taken from the glass bottles before they were sterilized. After pasteurization, the milk passes through microbiological quality control, and at this stage the pasteurized milk was sampled. Each sample was plated in duplicate onto plates with mannitol salt agar and incubated at 37C for 24 hours. The S. aureus isolates were evaluated by the disk diffusion methodology for sensitivity to erythromycin, tetracycline, clindamycin, oxacillin, ciprofloxacin, and ampicillin. The isolates were also tested against oxacillin and vancomycin by the microdilution method in Mueller–Hinton broth, as described in the Clinical and Laboratory Standards Institute.2 Methicillin-resistant S. aureus (MRSA) isolates were confirmed using conventional polymerase chain reaction (PCR).3 1 2 3
Moreover, the detection of MRSA in crude samples was also performed using real-time PCR methodology.4 The isolates were evaluated for their ability to produce biofilms by adhesion in microplates,5 and virulence genes in the studied staphylococci were subjected to conventional PCR.6 S. aureus was isolated from 29 (18%) of the total number of samples, from the raw milk it was recovered from 23 (29%) samples, and from glass bottles it was isolated from six (12%). S. aureus was not detected in samples of pasteurized milk (Table 1). Six of the 29 isolates of S. aureus were identified as resistant to oxacillin (minimum inhibitory concentration, ‡ 4 lg/mL). Five of these isolates were recovered from the raw milk, and one isolate was from the glass bottles. None was resistant to vancomycin (minimum inhibitory concentration, ‡ 16 lg/mL). In real-time PCR methodology, MRSA was characterized in approximately 89% (142/160) of samples. Regarding the analysis of microbial sensitivity by disk diffusion, methicillin-sensitive S. aureus isolates herein were particularly resistant to ampicillin, erythromycin, and tetracycline. However, all isolates were susceptible to vancomycin and clindamycin. The MRSA isolates were resistant to ampicillin, erythromycin, tetracycline, and ciprofloxacin, in addition to the already expected oxacillin resistance. All S. aureus isolates produced biofilm at different levels. Regarding the expression of virulence factors, the sec gene was detected in one (3%) isolate from a methicillinsensitive isolate recovered from raw milk. In Brazil, the frequency of S. aureus in human milk has varied among studies, but the frequency can range from 2.5% to 34%.1,7,8 Studies on the detection frequency of S. aureus in human milk in different countries have shown differences in frequency of detection varying widely between 2.5% and 100%.9,10 Human milk contaminated with MRSA additionally hampered control of infections caused by this bacterium in neonates or even in mothers. Studies on this issue are also scarce. Studies show that S. aureus strains, including resistant strains, can colonize human milk and serve as a source of infection for infants.11 Knowledge of the resistance profile of these strains can assist in implementing a more effective microbiological surveillance program.
State University of Santa Cruz, Ilhe´us, Bahia, Brazil. Federal University of Bahia, Vito´ria da Conquista, Bahia, Brazil. University of Sa˜o Paulo, Sa˜o Paulo, Brazil.
557
558
LETTER TO THE EDITOR
Table 1. Number of Isolates of S. aureus Using Culture and Methicillin-Resistant S. aureus Using the Microdilution Method Obtained from Samples Collected from a Brazilian Human Milk Bank in 2012 MRSA Isolation (culture (microdilution method)b Samples method)a
Breastfeeding Medicine 2014.9:557-558. Downloaded from online.liebertpub.com by ETH Zurich on 01/07/15. For personal use only.
Raw human milk Glass bottles Pasteurized human milk Total
80 50 30 160
23 6 0 29
(29) (12) (0) (18)
5 (22) 1 (17) — 6 (21)
Data are number (%). a Percentage relative to the total number of samples. b Percentage in relation to number of S. aureus isolates. MRSA, methicillin-resistant S. aureus.
The real-time PCR methodology was more sensitive than conventional PCR to detect MRSA isolates. In our study, there was a substantial difference in the detection of S. aureus in the samples analyzed by real-time PCR compared with a culture method, possibly related to the high sensitivity level of detection of the real-time PCR technology. However, realtime PCR measures gene fragments and not bacteria. Even in the absence of viable microorganisms in the sample, it is important to be very careful to avoid transmission of resistance genes in breastmilk. These resistance genes can be transferred to bacteria present in the microbiota of the infant. The presence of S. aureus with the sec gene in raw milk is important because the respective enterotoxin is resistant to heat treatment such as pasteurization and can be transmitted to premature infants.12 Another important pathogenic mechanism of S. aureus is biofilm production, which is an aspect on which few studies have been done on S. aureus strains isolated from human milk and vials. The strains isolated in this study were tested for biofilm formation, and the results showed that 100% of the strains produced biofilm. The recovery of S. aureus in human milk indicates poor hygiene and carelessness in milk storage procedures. Because of the important role this microorganism plays in infection in neonates, it should be included in the assessment of milk quality before and after pasteurization. Despite the presence of bacteria in raw milk, in the analysis of pasteurized milk samples, no growth of S. aureus was detected. The pasteurization was shown to be a good procedure to control this bacterium, irrespective of the status of the raw milk. The nation of Brazil has reduced its infant mortality rate using breastmilk. For this, the microbiological quality of human milk in Brazil used to feed neonates is well regulated. Acknowledgments
We wish to thank the Human Milk Bank of Esau´ Matos Municipal Hospital for allowing this study. This study was supported by the Programa de Apoio a Pesquisadores Emergentes da Universidade Federal da Bahia (PRODOC 02/ 2011). We thank Aricelma P. Franc¸a for invaluable technical assistance and AcademicEnglishSolutions.com for revising the English.
Disclosure Statement
No competing financial interests exist. References
1. Novak F, Almeida J, Warnken M, et al. Methicillin-resistant Staphylococcus aureus in human milk. Mem Inst Oswaldo Cruz 2000;95:29–33. 2. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; TwentySecond Informational Supplement. CLSI document M100S22. Wayne, PA: Clinical and Laboratory Standards Institute, 2012. 3. Perez-Roth E, Claverie-Martin F, Villar F, et al. Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance. J Clin Microbiol 2001;39:4037–4041. 4. Fosheim G, Nicholson A, Albrecht V, et al. Multiplex realtime PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes. J Clin Microbiol 2011;49:3071–3073. 5. Karaolis D, Rashid M, Chythanya R, et al. c-Di-GMP (3’5’-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation. Antimicrob Agents Chemother 2005;49:1029–1038. 6. Proietti P, Coppola G, Bietta A, et al. Characterization of genes encoding virulence determinants and toxins in Staphylococcus aureus from bovine milk in Central Italy. J Vet Med Sci 2010;72:1443–1448. 7. Pereira M, Santos E, Sellos I, et al. Staphylococci in breast milk from women without mastites. Rev Microbiol 1995; 26:117–120. 8. Silveira L, D’Amorimb M, Silva V, et al. Microbiological control of breast milk from a university hospital. Rev Baiana Saude Publ 2012;36:844–850. 9. Carroll L, Davies D, Osman M, et al. Bacteriological criteria for feeding raw breast-milk to babies on neonatal units. Lancet 1979;2:732–733. 10. Israel-Ballard K, Coutsoudis A, Chantry C, et al. Bacterial safety of flash-heated and unheated expressed breastmilk during storage. J Trop Pediatr 2006;52:399–405. 11. Barbe C, Santerne B, Lemartelleur L, et al. Prevalence of methicillin-resistant Staphylococcus aureus in expressed breast milk in a neonatal intensive care unit. J Hosp Infect 2008;69:195–197. 12. Carneiro L, Queiroz M, Merquior V. Antimicrobial-resistance and enterotoxin-encoding genes among staphylococci isolated from expressed human breast milk. J Med Microbiol 2004;53:761–768.
Address correspondence to: Lucas Miranda Marques, PhD Instituto Multidisciplinar em Sau´de Campus Anı´sio Teixeir Universidade Federal da Bahia Rua Rio de Contas, 58 Quadra 17, Lote 58 Bairro Candeias, CEP 45.029-094 Vito´ria da Conquista, Bahia, Brazil E-mail: [email protected]
Breastfeeding Medicine 2014.9:557-558. Downloaded from online.liebertpub.com by ETH Zurich on 01/07/15. For personal use only.
Detection, Antibiotic Resistance, and Pathogenicity of Staphylococci in Samples from a Brazilian Human Milk Bank Jessica Bomfim de Almeida,1,2 Suzi Pacheco de Carvalho,1 Andre´ Luı´s Vivas de Almeida,2 Guilherme Barreto Campos,3 Ma´rcio Vasconcelos Oliveira,2 Jorge Timenetsky,3 and Lucas Miranda Marques1,2
Dear Editor:
S
tudies on Staphylococcus aureus in samples of stored milk used to feed newborns are scarce in the literature.1 Therefore the aim of this study is to detect S. aureus in samples of raw milk, pasteurized milk, and glass bottles collected in a Brazilian human milk bank. The Human Milk Bank of Esau´ Matos Municipal Hospital is located in Vito´ria da Conquista, Bahia, Brazil. It was opened in 2004, when 22.5 L of milk was collected for newborns of the neonatal intensive care unit. Now the hospital has 682 donors providing about 878 L of milk per year. It is considered one of the largest milk banks in the region. All donated milk undergoes a pasteurization process in the service itself. The milking is done manually, and processing includes storing raw human milk in refrigerators in glass bottles previously sterilized by autoclaving, defrosting and rebottling, labeling of bottles, physicochemical quality control, pasteurization at 62.5C for 30 minutes, determining the microbiological quality of pasteurized human milk, and storage, distribution, and portioning. For the analysis, in total, 160 samples were collected. Of these, 80 samples were from raw human milk, 30 from pasteurized human milk, and 50 swabs from glass bottles for storing milk. Physicochemical quality control was performed on the samples. The samples were taken from the glass bottles before they were sterilized. After pasteurization, the milk passes through microbiological quality control, and at this stage the pasteurized milk was sampled. Each sample was plated in duplicate onto plates with mannitol salt agar and incubated at 37C for 24 hours. The S. aureus isolates were evaluated by the disk diffusion methodology for sensitivity to erythromycin, tetracycline, clindamycin, oxacillin, ciprofloxacin, and ampicillin. The isolates were also tested against oxacillin and vancomycin by the microdilution method in Mueller–Hinton broth, as described in the Clinical and Laboratory Standards Institute.2 Methicillin-resistant S. aureus (MRSA) isolates were confirmed using conventional polymerase chain reaction (PCR).3 1 2 3
Moreover, the detection of MRSA in crude samples was also performed using real-time PCR methodology.4 The isolates were evaluated for their ability to produce biofilms by adhesion in microplates,5 and virulence genes in the studied staphylococci were subjected to conventional PCR.6 S. aureus was isolated from 29 (18%) of the total number of samples, from the raw milk it was recovered from 23 (29%) samples, and from glass bottles it was isolated from six (12%). S. aureus was not detected in samples of pasteurized milk (Table 1). Six of the 29 isolates of S. aureus were identified as resistant to oxacillin (minimum inhibitory concentration, ‡ 4 lg/mL). Five of these isolates were recovered from the raw milk, and one isolate was from the glass bottles. None was resistant to vancomycin (minimum inhibitory concentration, ‡ 16 lg/mL). In real-time PCR methodology, MRSA was characterized in approximately 89% (142/160) of samples. Regarding the analysis of microbial sensitivity by disk diffusion, methicillin-sensitive S. aureus isolates herein were particularly resistant to ampicillin, erythromycin, and tetracycline. However, all isolates were susceptible to vancomycin and clindamycin. The MRSA isolates were resistant to ampicillin, erythromycin, tetracycline, and ciprofloxacin, in addition to the already expected oxacillin resistance. All S. aureus isolates produced biofilm at different levels. Regarding the expression of virulence factors, the sec gene was detected in one (3%) isolate from a methicillinsensitive isolate recovered from raw milk. In Brazil, the frequency of S. aureus in human milk has varied among studies, but the frequency can range from 2.5% to 34%.1,7,8 Studies on the detection frequency of S. aureus in human milk in different countries have shown differences in frequency of detection varying widely between 2.5% and 100%.9,10 Human milk contaminated with MRSA additionally hampered control of infections caused by this bacterium in neonates or even in mothers. Studies on this issue are also scarce. Studies show that S. aureus strains, including resistant strains, can colonize human milk and serve as a source of infection for infants.11 Knowledge of the resistance profile of these strains can assist in implementing a more effective microbiological surveillance program.
State University of Santa Cruz, Ilhe´us, Bahia, Brazil. Federal University of Bahia, Vito´ria da Conquista, Bahia, Brazil. University of Sa˜o Paulo, Sa˜o Paulo, Brazil.
557
558
LETTER TO THE EDITOR
Table 1. Number of Isolates of S. aureus Using Culture and Methicillin-Resistant S. aureus Using the Microdilution Method Obtained from Samples Collected from a Brazilian Human Milk Bank in 2012 MRSA Isolation (culture (microdilution method)b Samples method)a
Breastfeeding Medicine 2014.9:557-558. Downloaded from online.liebertpub.com by ETH Zurich on 01/07/15. For personal use only.
Raw human milk Glass bottles Pasteurized human milk Total
80 50 30 160
23 6 0 29
(29) (12) (0) (18)
5 (22) 1 (17) — 6 (21)
Data are number (%). a Percentage relative to the total number of samples. b Percentage in relation to number of S. aureus isolates. MRSA, methicillin-resistant S. aureus.
The real-time PCR methodology was more sensitive than conventional PCR to detect MRSA isolates. In our study, there was a substantial difference in the detection of S. aureus in the samples analyzed by real-time PCR compared with a culture method, possibly related to the high sensitivity level of detection of the real-time PCR technology. However, realtime PCR measures gene fragments and not bacteria. Even in the absence of viable microorganisms in the sample, it is important to be very careful to avoid transmission of resistance genes in breastmilk. These resistance genes can be transferred to bacteria present in the microbiota of the infant. The presence of S. aureus with the sec gene in raw milk is important because the respective enterotoxin is resistant to heat treatment such as pasteurization and can be transmitted to premature infants.12 Another important pathogenic mechanism of S. aureus is biofilm production, which is an aspect on which few studies have been done on S. aureus strains isolated from human milk and vials. The strains isolated in this study were tested for biofilm formation, and the results showed that 100% of the strains produced biofilm. The recovery of S. aureus in human milk indicates poor hygiene and carelessness in milk storage procedures. Because of the important role this microorganism plays in infection in neonates, it should be included in the assessment of milk quality before and after pasteurization. Despite the presence of bacteria in raw milk, in the analysis of pasteurized milk samples, no growth of S. aureus was detected. The pasteurization was shown to be a good procedure to control this bacterium, irrespective of the status of the raw milk. The nation of Brazil has reduced its infant mortality rate using breastmilk. For this, the microbiological quality of human milk in Brazil used to feed neonates is well regulated. Acknowledgments
We wish to thank the Human Milk Bank of Esau´ Matos Municipal Hospital for allowing this study. This study was supported by the Programa de Apoio a Pesquisadores Emergentes da Universidade Federal da Bahia (PRODOC 02/ 2011). We thank Aricelma P. Franc¸a for invaluable technical assistance and AcademicEnglishSolutions.com for revising the English.
Disclosure Statement
No competing financial interests exist. References
1. Novak F, Almeida J, Warnken M, et al. Methicillin-resistant Staphylococcus aureus in human milk. Mem Inst Oswaldo Cruz 2000;95:29–33. 2. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; TwentySecond Informational Supplement. CLSI document M100S22. Wayne, PA: Clinical and Laboratory Standards Institute, 2012. 3. Perez-Roth E, Claverie-Martin F, Villar F, et al. Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance. J Clin Microbiol 2001;39:4037–4041. 4. Fosheim G, Nicholson A, Albrecht V, et al. Multiplex realtime PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes. J Clin Microbiol 2011;49:3071–3073. 5. Karaolis D, Rashid M, Chythanya R, et al. c-Di-GMP (3’5’-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation. Antimicrob Agents Chemother 2005;49:1029–1038. 6. Proietti P, Coppola G, Bietta A, et al. Characterization of genes encoding virulence determinants and toxins in Staphylococcus aureus from bovine milk in Central Italy. J Vet Med Sci 2010;72:1443–1448. 7. Pereira M, Santos E, Sellos I, et al. Staphylococci in breast milk from women without mastites. Rev Microbiol 1995; 26:117–120. 8. Silveira L, D’Amorimb M, Silva V, et al. Microbiological control of breast milk from a university hospital. Rev Baiana Saude Publ 2012;36:844–850. 9. Carroll L, Davies D, Osman M, et al. Bacteriological criteria for feeding raw breast-milk to babies on neonatal units. Lancet 1979;2:732–733. 10. Israel-Ballard K, Coutsoudis A, Chantry C, et al. Bacterial safety of flash-heated and unheated expressed breastmilk during storage. J Trop Pediatr 2006;52:399–405. 11. Barbe C, Santerne B, Lemartelleur L, et al. Prevalence of methicillin-resistant Staphylococcus aureus in expressed breast milk in a neonatal intensive care unit. J Hosp Infect 2008;69:195–197. 12. Carneiro L, Queiroz M, Merquior V. Antimicrobial-resistance and enterotoxin-encoding genes among staphylococci isolated from expressed human breast milk. J Med Microbiol 2004;53:761–768.
Address correspondence to: Lucas Miranda Marques, PhD Instituto Multidisciplinar em Sau´de Campus Anı´sio Teixeir Universidade Federal da Bahia Rua Rio de Contas, 58 Quadra 17, Lote 58 Bairro Candeias, CEP 45.029-094 Vito´ria da Conquista, Bahia, Brazil E-mail: [email protected]
