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A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition
Journal of Hospital Infection (2004) 58, 122–127
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A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition D.M. Matsuokaa, S.F. Costaa,c, C. Manginia, G.M.D. Almeidab, C.N. Bentob, ´voraa, I.M. Van Der Heijdenc, R.E. Soaresc, S. Gobaraa, L.G.F. Ta a,c,d, * A.S. Levin a
˜o Paulo, Sa ˜o Paulo, Brazil Nosocomial Infection Control Group, Hospital das Clı´nicas, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil Laboratory of Microbiology, Hospital das Clı´nicas, University of Sa c ˜o Paulo, Sa ˜o Paulo, Laboratory of Medical Investigation (LIM-54), Hospital das Clı´nicas, University of Sa Brazil d ˜o Paulo, Sa ˜o Paulo, Brazil Department of Infectious Diseases, Hospital das Clı´nicas, University of Sa b
Received 25 August 2003; accepted 25 February 2004
KEYWORDS Salmonella enteritidis; Nosocomial outbreak; Enteral nutrition; Lyophilization
Summary Outbreaks of Salmonella spp. gastro-enteritis in hospitals are of concern because of the increased susceptibility of patients and associated high morbidity. This study is a report of a nosocomial outbreak of Salmonella enteritidis associated with enteral nutrition. In December 1999, one sample of enteral feed tested positive for S. enteritidis. During the subsequent 6 weeks, eight cases of nosocomial salmonellosis occurred. Patients involved in the outbreak were aged 19 –79 years (median ¼ 36.5), and salmonella was isolated from the blood of two patients. All patients were receiving enteral nutrition at the time and all had diarrhoea. Three patients died. All 13 employees of the Nutrition Department were asymptomatic and their stool samples were negative. Environmental and water samples were also negative. The diet, however, contained lyophilized egg albumin. Molecular typing showed that the isolates of seven patients were indistinguishable from the one obtained from the enteral diet. It was thought that the nosocomial salmonellosis probably occurred due to the use of a commercial lyophilized diet. Another method of processing diets may be necessary to ensure patient safety. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction *Corresponding author. Address: Department of Infectious Diseases, Hospital das Clı´nicas, University of Sa ˜o Paulo, Rua Harmonia, 564/52, Sa ˜o Paulo SP 05435-000, Brazil. Tel./fax: þ 55-11-3069-7066. E-mail address: [email protected]
Salmonella is a common cause of food-borne outbreaks and infection is usually acquired by ingestion of contaminated food items, such as meat, poultry, eggs, milk and vegetables.1 Salmonella enteritidis
0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.05.003
A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition can be found inside eggs,2 and should the eggs be eaten raw or undercooked, it can cause illness. Contamination offood by the hands offood handlers is unusual if proper hygiene is practised,3 – 5 but when this is breached, outbreaks can also occur.6 Outbreaks of salmonellosis in hospitals are of special concern as there is an increased susceptibility of patients, with an associated morbidity. Outbreaks and their control measures can seriously disrupt hospital services.7,8 Nosocomial transmission of salmonella during outbreaks has been reported occasionally, usually resulting from person-to-person spread.9 – 12 The purpose of this study was to report a nosocomial outbreak of S. enteritidis associated with enteral nutrition that occurred in a tertiary-care hospital.
Method The outbreak occurred in the Hospital das Clı´nicas of the University of Sa ˜o Paulo, Brazil, a tertiarycare university hospital with 1000 beds. The Nutrition and Dietary Department sends samples of enteral nutrition for microbiological examination on a weekly basis. A 200 mL bottle of the enteral diet is chosen at random and the sample is submitted to: (1) pre-enrichment procedure with buffered peptoned water 1% for 18 – 24 h at 35 8C; (2) selective enrichment procedure with selenite – cistine broth and modified Rappaport – Vasiliadis broth for 48 h at 42 8C; and (3) culture on plates of Salmonella – Shigella agar (SS agar), brilliant green agar and bismuth sulphate agar for 24 – 48 h at 35 8C. If colonies suspected of being Salmonella spp. are isolated, they are cultured in Adolfo Lutz Institute medium13 for 24 h at 35 8C and then tested for reactivity with Salmonella spp. antiserum.14 In December 1999, one bottle of enteral nutrition tested positive for S. enteritidis. During the period from 14 December 1999 to 28 January 2000, eight cases of nosocomial salmonellosis were detected. A case was defined as a patient who had Salmonella spp. isolated from a stool or blood culture, 48 h or more after hospitalization. Seven clinical samples positive for nosocomial Salmonella spp., the diet sample, one unrelated isolate from a stool culture from another hospital of another city (Carapicuiba) and one community strain obtained at the Hospital das Clı´nicas were submitted for molecular characterization using pulsed-field gel electrophoresis (PFGE). The preparation of chromosomal DNA for PFGE was performed as described elsewhere.15 Bacterial isolates were grown on blood agar overnight at 37 8C. Gel blocks were made by use of equal volumes of 2%
123
low-melting-point agar (Gibco) and a bacterial suspension of 9 £ 108 cells. DNA blocks were digested with two enzymes: SpeI and XbaI (New England bioLabs) at 37 8C for 12 h. PFGE was performed with use of 1% agarose gel (Gibco) in a CHEF DRII system (Bio-Rad) under the following conditions: run time 21 h; temperature 14 8C; 200 V; initial forward time 5 s; final forward time 35 s for SpeI; and initial forward time 1 s and final forward time 35 s for XbaI. Lambda concatamers were run in the first and last lanes. Gels were stained with ethidium bromide, washed in water and photographed under ultraviolet light. Strains isolated from stool and blood during the outbreak, the unrelated strains and the diet sample were compared. Genotypes were defined based on DNA banding patterns, and isolates with identical patterns were regarded as ‘genotypically indistinguishable’.16 During the outbreak investigation, enteral nutrition preparation, storage and handling procedures were reviewed by the Infection Control Department. Diet composition was evaluated based on the manufacturer’s information. All the employees of the Nutrition and Dietary Department submitted stools and hand swabs to investigate for micro-organisms, helminths and protozoa; the samples were cultured in selenite-F broth, SS agar and MacConkey agar for 24 h at 37 8C. Environmental samples (tubs, sinks and taps) were collected with cotton-tipped swabs, incubated on trypticase soya broth (TSB) at 37 8C for seven days and then cultured on SS agar, chocolate agar and MacConkey agar for 24 h at 37 8C. Retrospective data on the affected patients were collected by review of the medical records and included: age, sex, unit, underlying diseases, the use of enteral nutrition, presence or absence of diarrhoea, duration of hospitalization and the patients’ outcomes. The antibiotic susceptibility of all Salmonella spp. isolates was determined by the Kirby– Bauer method17 for ampicillin, ciprofloxacin, chloramphenicol, ceftriaxone, cefalotin, cefoxitin and gentamicin, and minimal inhibitory concentration (MIC) determination using the microdilution method was performed for ampicillin, ciprofloxacin, ceftriaxone, cefazolin, cefoxitin, gentamicin and trimethoprim/sulfamethoxazole.
Results Between 14 December 1999 and 28 January 2000, nine cases of salmonella infection were reported. Eight were considered to be hospital-acquired. One patient was excluded because he had a positive
Blood Stool Stool Stool Blood Stool Stool Stool þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ ICU, intensive care unit; þ , present.
22 27 110 16 12 39 70 30 Surgery (10th) Liver transplantation and infective endocarditis ICU Neurology (5th) Meningioma and sepsis Neurosurgery (5th) Atrial fibrillation and haemorrhagic cerebrovascular disease Rheumatology (8th) Cerebral palsy and systemic lupus erythematosus ICU Infectious diseases (4th) Staphylococcus aureus sepsis Nephrology/Renal transplant (7th) Haemolytic uraemic syndrome ICU Neurology (5th) Myopathy and pneumonia ICU Trauma (4th) Anti-phospholipid syndrome, abortion and sepsis 67 63 79 30 30 19 43 27 Male Male Male Female Male Female Male Female 1 2 3 4 5 6 7 8
Underlying diseases Unit (floor) Age (years) Sex Patient no
Table I Characteristics of the eight nosocomial cases of salmonellosis, Hospital das Clı´nicas, University of Sa ˜o Paulo, Brazil
blood culture for Salmonella spp. on hospital admission. In 1998 just two cases of salmonellosis had been reported. Of the eight patients with hospital-acquired salmonellosis, three were female. All the patients had received enteral nutrition and developed diarrhoea; Ages ranged from 19 to 79 years (median 36.5). Two patients had Salmonella spp. isolated from their blood. The patients were hospitalized in seven units, located on different floors. Four were in intensive care units, two in surgical units and two in medical units. Underlying diseases varied and are listed in Table I. Three patients died, 6, 25 and 133 days after developing the infection. The review of diet preparation showed that it occurred in an adequate location by employees using head coverings, masks, shoe covers and a specific uniform. Handwashing was done at a sink with photosensitive taps and with povidone –iodine containing 7.5% of free iodine in a detergent base and individual brushes for each employee. Instruments used for diet preparation were washed adequately at the beginning of the procedure. After the first production cycle, instruments were cleaned with water and detergent. The blender used was washed with detergent and rinsed with water. Buckets were cleaned with water and detergent, and disinfected with 70% alcohol at the beginning and the end of the procedures. The diet was sourced commercially and came as a powder in single-use, aluminium, sealed packages. The water used for dilution was provided via a filter fixed on the wall. The bottles, used to receive the enteral nutrition, were received as single packed units and filling was made via gravity. The bottles were then sealed mechanically. Distribution to the patient units was made in trays and the mechanically sealed bottles were covered with sterile drapes. All 13 employees of the Nutrition Department were asymptomatic and their cultures were negative for Salmonella spp., as were the samples obtained from tubs, taps and water. According to the manufacturer’s information, the enteral diet contained malthodextrin, soya extract, coconut oil, lyophilized albumin, corn oil, soya lecitin, vitamins and minerals. All strains of Salmonella spp. were susceptible to the tested antibiotics. PFGE showed that all the isolates from the outbreak patients and the one from the diet were genotypically indistinguishable. The Salmonella spp. sample from another hospital, located in another city, was unrelated to the outbreak strain, and the community sample from the same city was indistinguishable from the outbreak strain (Figure 1).
D.M. Matsuoka et al.
Duration of Enteral Diarrhoea Culture hospitalization nutrition use before Salmonella isolated (days)
124
A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition
125
Figure 1 Molecular typing by pulsed-field gel electrophoresis of salmonella isolates. Lanes 1: molecular marker; Lane 2: ATCC 14028 Salmonella typhi; Lanes 3 to 9: nosocomial Salmonella Enteritidis isolates; Lane 10: community isolate from the city of Sa ˜o Paulo; Lane 11: community isolate from another city; Lane 12: isolate from the enteral diet.
Discussion Nosocomial outbreaks of Salmonella spp. infection are not uncommon and most hospital outbreaks are small and last days or weeks.3,18 Predisposing host risk factors may facilitate infection by smaller inocula of Salmonella spp., such as the prior use of antibiotics, antacids or H2 antagonists, presence of malignancy, liver disease, haemoglobinopathies, gastrectomy or achlorhydria.19,20 The age of the host is also an important determinant of susceptibility to infection. Children aged less than one year and people over 60 years acquire salmonellosis more frequently than other age groups, and the severity of illness is greater.19,21,22 Such patients may become bacteraemic. In our patients, prior antibiotic use, underlying liver disease and malignancy may have contributed to diarrhoea. Three patients died, but death was not related to salmonellosis, according to their medical records. The vehicle of transmission of Salmonella spp. in this outbreak was considered to be the enteral diet. Salmonellosis associated with enteral nutrition, including typhoid organisms, has not been reported previously. After the detection of a sample of enteral diet as being positive for salmonella, the Infection Control Department searched for nosocomial cases of salmonellosis. As a result, eight cases were identified. The patients were hospitalized in different
units and their underlying diseases were diverse. On review of the medical records, it was found that all patients had received enteral nutrition. The Nutrition and Dietary Department was therefore visited and enteral nutrition preparation, storage and handling procedures were reviewed. No problems were found with the employees or with the diet preparation procedures. All cultures of tubs, taps and filtered water were negative. These data suggested that diet was probably contaminated before re-hydration. In our study, all isolates of Salmonella spp. (clinical and diet samples) were indistinguishable and different from the strain from another city (Carapicuiba), using PFGE typing. One isolate detected on admission, in a blood culture sample from a patient unrelated to the outbreak, was genotypically indistinguishable from the nosocomial strains. This patient had not been hospitalized previously, had not received an enteral diet, and had had no contact with the nosocomial cases. We concluded therefore that in this outbreak, the Salmonella spp. in the nosocomial environment originated from a community source connected to the Salmonella serovars found in the city of Sa ˜o Paulo. Tavechio et al.23 showed an increase in S. enteritidis infection in the State of Sa ˜o Paulo, Brazil, associated with the consumption of raw eggs, as indicated by epidemiological investigation
126
of outbreaks. In the late 1970s, strains of S. typhimurium were mainly associated with nosocomial infections in Sa ˜o Paulo.23 – 25 The diet powder contained lyophilized albumin extracted from eggs that could have been the source of salmonella infection. Vaccination of chickens may make egg production safer,26 but in Brazil vaccination of flocks is minimal. There are no regulations, and public awareness about the problem and pressure for control is non-existent. Lyophilization or freeze-drying is a method used for food conservation in which water is removed by sublimation. The material is frozen at 2 40 8F then is heated to very high temperatures. As a result, humidity contained in the food goes directly from the solid to the gaseous state. The procedures and reasons for using lyophilization are varied.27 The effects of lyophilization on the viability and storage of attenuated live microbial vaccines were studied for Salmonella, Shigella and Escherichia coli, and lyophilization was found to maintain the biological, antigenic and genetic characteristics of the fresh microorganisms.28 Bergmann et al.29 showed that lyophilization does not destroy cells. Electron microscopy showed that the process only decreased diameter and caused the cell wall to become wavy, whilst the basic structures of cells remained intact. Sinskey et al.30 showed that freezing at 2 40 8 F did not decrease the viable population of S. typhimurium below that of the initial population. Simon et al.31 did not observe any alteration in virulence of S. typhimurium after lyophilization, although organisms metabolically injured by freezing may have slower growth rates. All these data suggest that the egg albumin in the enteral diets may have been the cause of the outbreak and that lyophilization can be used for food preservation, but to assure sterilization, other methods should be employed.
References 1. Khuri-Bulos NA, Abu Khalaf M, Shehabi A, Shami K. Foodhandler-associated salmonella outbreak in a university hospital despite routine surveillance cultures of kitchen employees. Infect Control Hosp Epidemiol 1994;15: 311—314. 2. Telzak EE, Budnick LD, Greenberg MSZ, et al. A nosocomial outbreak of Salmonella enteritidis infection due to consumption of raw eggs. N Engl J Med 1990;323:394—397. 3. Dryden MS, Keyworth N, Gabb R, Stein K. Asymptomatic food handlers as the source of nosocomial salmonellosis. J Hosp Infect 1994;28:195—208. 4. McCall B, McCormack JG, Stafford R, Towner C. An outbreak of Salmonella typhimurium at a teaching hospital. Infect Control Hosp Epidemiol 1999;20:55—56. 5. Nathwani D, Wood WJ. Ciprofloxacin and salmonella carriage. J Hosp Infect 1992;22:181—184.
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6. Opal SM, Mayer KH, Roland F, Brondum J, Heelan J, Lyhte L. Investigation of a foodborne outbreak of salmonellosis among hospital employees. Am J Infect Control 1989;17: 141—147. 7. Joseph CA, Palmer SR. Outbreaks of salmonella infection in hospitals in England and Wales, 1978—87. BMJ 1989;298: 1161—1164. 8. Kumarasinghe G, Hamilton WJ, Gould JD, Palmer SR, Dudgeon JA, Marshall WC. An outbreak of Salmonella muenchen infection in a specialist paediatric hospital. J Hosp Infect 1982;3:341—344. 9. Weikel CS, Guerrant RL. Nosocomial salmonellosis. Infect Control 1985;6:218—220. 10. Choi M, Yoshikawa TT, Bridge J, et al. Salmonella outbreak in a nursing home. J Am Geriatr Soc 1990;38:531—534. 11. Schroeder SA, Aserkoff B, Brachman PS. Epidemic salmonellosis in hospitals and institutions: a five-year review. N Engl J Med 1968;279:674—678. 12. Steere AC, Hall 3rd WJ, Wells JG, et al. Person-to-person spread of Salmonella typhimurium after a hospital commonsource outbreak. Lancet 1975;1:319—322. 13. Pessoa GVA, Silva EAM. Me ´dium of Rugai and lysine-motility combined in one single tube for presumptive identification of Enterobacteriaceae. Rev Inst Adolfo Lutz 1972;32: 97—100. ´niques des 14. Popoff MY, Le Minor L, editors. Formules antige ´rovars de Salmonella. Paris: Centre Collaborateur OMS de se Re ´ference et de Recherches pour les Salmonella; 1997. p. 151. 15. Pfaller MA. Chromosomal restriction fragment analysis by pulsed-field gel electrophoresis. In: Isemberg HD, editor. Clinical microbiology procedure handbook. Washington, DC: American Society for Microbiology; 1993. p. 10.5.C.1-10. 5C.11. 16. Tenover FC, Arbeit RD, Goering RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Infect Control Hosp Epidemiol 1997;18: 426—439. 17. Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493—496. 18. Palmer SR, Rowe B. Investigation of salmonella in hospitals. BMJ 1983;287:891—893. 19. Riley LW, Cohen ML, Seals JE, et al. Importance of host factors in human salmonellosis caused by multiresistant strains of salmonella. J Infect Dis 1984;149:878—883. 20. Blaser MJ, Newman LS. A review of human salmonellosis. I: Infective dose. Rev Infect Dis 1982;4:1096—1106. 21. Standaert SM, Hutcheson RH, Schaffner W. Nosocomial transmission of salmonella gastroenteritis to laundry workers in a nursing home. Infect Control Hosp Epidemiol 1994;15:22—26. 22. Blaser MJ, Feldman RA. Salmonella bacteremia: reports to the Centers for Disease Control 1968—1979. J Infect Dis 1981;143:743—746. 23. Tavechio AT, Fernandes SA, Neves BC, Dias AMG, Irino K. Changing patterns of Salmonella serovars: increase of Salmonella enteritidis in Sa ˜o Paulo, Brazil. Rev Inst Med Trop Sao Paulo 1996;38:315—322. 24. Pessoa GVA, Irino K, Melles CE. Ocorre ˆncia de bacte ´rias enteropatoge ˆnicas em Sa ˜o Paulo no septie ˆnio 1970—76. I: sorotipos de Salmonella isolados e identificados. Rev Inst Adolfo Lutz 1978;38:107—127. 25. Taunay AE, Fernandes SA, Tavechio AT, Neves BC, Dias AM, Irino L. The role of public health laboratory in the problem of
A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition
salmonellosis in Sa ˜o Paulo, Brazil. Rev Inst Med Trop Sao Paulo 1996;38:119—127. 26. Davies R, Breslin M. Effects of vaccination and other preventive methods for Salmonella enteritidis on commercial laying chicken farms. Vet Rec 2003;153:673—677. 27. Rhoades HE. Effects of 20 years’ storage on lyophilized cultures of bacteria, molds, viruses and yeasts. Am J Vet Res 1970;31:1867—1870. 28. Dima VF, Vasilescu T. Biologic properties of lyophilized live typhoid vaccine strains. Arch Roum Path Exp Microbiol 1979; 38:213—219.
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29. Bergmann P, Mebel S, Rustenbach S. Morphology of Salmonella typhi in different inactivation techniques. Acta Microbiol Acad Sci Hung 1977;24:207—219. 30. Sinskey TJ, McIntosh AH, Pablo IS, Silverman GJ, Goldblith SA. Considerations in the recovery of micro-organisms from freeze-dried foods. Health Lab Sci 1964;1:297—306. 31. Simon EM, Stahl KL, Wilson JB. Preservation by freeze-drying and the stability of virulence of Salmonella typhimurium. Appl Microbiol 1963;11:371—376.
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A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition D.M. Matsuokaa, S.F. Costaa,c, C. Manginia, G.M.D. Almeidab, C.N. Bentob, ´voraa, I.M. Van Der Heijdenc, R.E. Soaresc, S. Gobaraa, L.G.F. Ta a,c,d, * A.S. Levin a
˜o Paulo, Sa ˜o Paulo, Brazil Nosocomial Infection Control Group, Hospital das Clı´nicas, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil Laboratory of Microbiology, Hospital das Clı´nicas, University of Sa c ˜o Paulo, Sa ˜o Paulo, Laboratory of Medical Investigation (LIM-54), Hospital das Clı´nicas, University of Sa Brazil d ˜o Paulo, Sa ˜o Paulo, Brazil Department of Infectious Diseases, Hospital das Clı´nicas, University of Sa b
Received 25 August 2003; accepted 25 February 2004
KEYWORDS Salmonella enteritidis; Nosocomial outbreak; Enteral nutrition; Lyophilization
Summary Outbreaks of Salmonella spp. gastro-enteritis in hospitals are of concern because of the increased susceptibility of patients and associated high morbidity. This study is a report of a nosocomial outbreak of Salmonella enteritidis associated with enteral nutrition. In December 1999, one sample of enteral feed tested positive for S. enteritidis. During the subsequent 6 weeks, eight cases of nosocomial salmonellosis occurred. Patients involved in the outbreak were aged 19 –79 years (median ¼ 36.5), and salmonella was isolated from the blood of two patients. All patients were receiving enteral nutrition at the time and all had diarrhoea. Three patients died. All 13 employees of the Nutrition Department were asymptomatic and their stool samples were negative. Environmental and water samples were also negative. The diet, however, contained lyophilized egg albumin. Molecular typing showed that the isolates of seven patients were indistinguishable from the one obtained from the enteral diet. It was thought that the nosocomial salmonellosis probably occurred due to the use of a commercial lyophilized diet. Another method of processing diets may be necessary to ensure patient safety. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction *Corresponding author. Address: Department of Infectious Diseases, Hospital das Clı´nicas, University of Sa ˜o Paulo, Rua Harmonia, 564/52, Sa ˜o Paulo SP 05435-000, Brazil. Tel./fax: þ 55-11-3069-7066. E-mail address: [email protected]
Salmonella is a common cause of food-borne outbreaks and infection is usually acquired by ingestion of contaminated food items, such as meat, poultry, eggs, milk and vegetables.1 Salmonella enteritidis
0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.05.003
A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition can be found inside eggs,2 and should the eggs be eaten raw or undercooked, it can cause illness. Contamination offood by the hands offood handlers is unusual if proper hygiene is practised,3 – 5 but when this is breached, outbreaks can also occur.6 Outbreaks of salmonellosis in hospitals are of special concern as there is an increased susceptibility of patients, with an associated morbidity. Outbreaks and their control measures can seriously disrupt hospital services.7,8 Nosocomial transmission of salmonella during outbreaks has been reported occasionally, usually resulting from person-to-person spread.9 – 12 The purpose of this study was to report a nosocomial outbreak of S. enteritidis associated with enteral nutrition that occurred in a tertiary-care hospital.
Method The outbreak occurred in the Hospital das Clı´nicas of the University of Sa ˜o Paulo, Brazil, a tertiarycare university hospital with 1000 beds. The Nutrition and Dietary Department sends samples of enteral nutrition for microbiological examination on a weekly basis. A 200 mL bottle of the enteral diet is chosen at random and the sample is submitted to: (1) pre-enrichment procedure with buffered peptoned water 1% for 18 – 24 h at 35 8C; (2) selective enrichment procedure with selenite – cistine broth and modified Rappaport – Vasiliadis broth for 48 h at 42 8C; and (3) culture on plates of Salmonella – Shigella agar (SS agar), brilliant green agar and bismuth sulphate agar for 24 – 48 h at 35 8C. If colonies suspected of being Salmonella spp. are isolated, they are cultured in Adolfo Lutz Institute medium13 for 24 h at 35 8C and then tested for reactivity with Salmonella spp. antiserum.14 In December 1999, one bottle of enteral nutrition tested positive for S. enteritidis. During the period from 14 December 1999 to 28 January 2000, eight cases of nosocomial salmonellosis were detected. A case was defined as a patient who had Salmonella spp. isolated from a stool or blood culture, 48 h or more after hospitalization. Seven clinical samples positive for nosocomial Salmonella spp., the diet sample, one unrelated isolate from a stool culture from another hospital of another city (Carapicuiba) and one community strain obtained at the Hospital das Clı´nicas were submitted for molecular characterization using pulsed-field gel electrophoresis (PFGE). The preparation of chromosomal DNA for PFGE was performed as described elsewhere.15 Bacterial isolates were grown on blood agar overnight at 37 8C. Gel blocks were made by use of equal volumes of 2%
123
low-melting-point agar (Gibco) and a bacterial suspension of 9 £ 108 cells. DNA blocks were digested with two enzymes: SpeI and XbaI (New England bioLabs) at 37 8C for 12 h. PFGE was performed with use of 1% agarose gel (Gibco) in a CHEF DRII system (Bio-Rad) under the following conditions: run time 21 h; temperature 14 8C; 200 V; initial forward time 5 s; final forward time 35 s for SpeI; and initial forward time 1 s and final forward time 35 s for XbaI. Lambda concatamers were run in the first and last lanes. Gels were stained with ethidium bromide, washed in water and photographed under ultraviolet light. Strains isolated from stool and blood during the outbreak, the unrelated strains and the diet sample were compared. Genotypes were defined based on DNA banding patterns, and isolates with identical patterns were regarded as ‘genotypically indistinguishable’.16 During the outbreak investigation, enteral nutrition preparation, storage and handling procedures were reviewed by the Infection Control Department. Diet composition was evaluated based on the manufacturer’s information. All the employees of the Nutrition and Dietary Department submitted stools and hand swabs to investigate for micro-organisms, helminths and protozoa; the samples were cultured in selenite-F broth, SS agar and MacConkey agar for 24 h at 37 8C. Environmental samples (tubs, sinks and taps) were collected with cotton-tipped swabs, incubated on trypticase soya broth (TSB) at 37 8C for seven days and then cultured on SS agar, chocolate agar and MacConkey agar for 24 h at 37 8C. Retrospective data on the affected patients were collected by review of the medical records and included: age, sex, unit, underlying diseases, the use of enteral nutrition, presence or absence of diarrhoea, duration of hospitalization and the patients’ outcomes. The antibiotic susceptibility of all Salmonella spp. isolates was determined by the Kirby– Bauer method17 for ampicillin, ciprofloxacin, chloramphenicol, ceftriaxone, cefalotin, cefoxitin and gentamicin, and minimal inhibitory concentration (MIC) determination using the microdilution method was performed for ampicillin, ciprofloxacin, ceftriaxone, cefazolin, cefoxitin, gentamicin and trimethoprim/sulfamethoxazole.
Results Between 14 December 1999 and 28 January 2000, nine cases of salmonella infection were reported. Eight were considered to be hospital-acquired. One patient was excluded because he had a positive
Blood Stool Stool Stool Blood Stool Stool Stool þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ ICU, intensive care unit; þ , present.
22 27 110 16 12 39 70 30 Surgery (10th) Liver transplantation and infective endocarditis ICU Neurology (5th) Meningioma and sepsis Neurosurgery (5th) Atrial fibrillation and haemorrhagic cerebrovascular disease Rheumatology (8th) Cerebral palsy and systemic lupus erythematosus ICU Infectious diseases (4th) Staphylococcus aureus sepsis Nephrology/Renal transplant (7th) Haemolytic uraemic syndrome ICU Neurology (5th) Myopathy and pneumonia ICU Trauma (4th) Anti-phospholipid syndrome, abortion and sepsis 67 63 79 30 30 19 43 27 Male Male Male Female Male Female Male Female 1 2 3 4 5 6 7 8
Underlying diseases Unit (floor) Age (years) Sex Patient no
Table I Characteristics of the eight nosocomial cases of salmonellosis, Hospital das Clı´nicas, University of Sa ˜o Paulo, Brazil
blood culture for Salmonella spp. on hospital admission. In 1998 just two cases of salmonellosis had been reported. Of the eight patients with hospital-acquired salmonellosis, three were female. All the patients had received enteral nutrition and developed diarrhoea; Ages ranged from 19 to 79 years (median 36.5). Two patients had Salmonella spp. isolated from their blood. The patients were hospitalized in seven units, located on different floors. Four were in intensive care units, two in surgical units and two in medical units. Underlying diseases varied and are listed in Table I. Three patients died, 6, 25 and 133 days after developing the infection. The review of diet preparation showed that it occurred in an adequate location by employees using head coverings, masks, shoe covers and a specific uniform. Handwashing was done at a sink with photosensitive taps and with povidone –iodine containing 7.5% of free iodine in a detergent base and individual brushes for each employee. Instruments used for diet preparation were washed adequately at the beginning of the procedure. After the first production cycle, instruments were cleaned with water and detergent. The blender used was washed with detergent and rinsed with water. Buckets were cleaned with water and detergent, and disinfected with 70% alcohol at the beginning and the end of the procedures. The diet was sourced commercially and came as a powder in single-use, aluminium, sealed packages. The water used for dilution was provided via a filter fixed on the wall. The bottles, used to receive the enteral nutrition, were received as single packed units and filling was made via gravity. The bottles were then sealed mechanically. Distribution to the patient units was made in trays and the mechanically sealed bottles were covered with sterile drapes. All 13 employees of the Nutrition Department were asymptomatic and their cultures were negative for Salmonella spp., as were the samples obtained from tubs, taps and water. According to the manufacturer’s information, the enteral diet contained malthodextrin, soya extract, coconut oil, lyophilized albumin, corn oil, soya lecitin, vitamins and minerals. All strains of Salmonella spp. were susceptible to the tested antibiotics. PFGE showed that all the isolates from the outbreak patients and the one from the diet were genotypically indistinguishable. The Salmonella spp. sample from another hospital, located in another city, was unrelated to the outbreak strain, and the community sample from the same city was indistinguishable from the outbreak strain (Figure 1).
D.M. Matsuoka et al.
Duration of Enteral Diarrhoea Culture hospitalization nutrition use before Salmonella isolated (days)
124
A nosocomial outbreak of Salmonella enteritidis associated with lyophilized enteral nutrition
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Figure 1 Molecular typing by pulsed-field gel electrophoresis of salmonella isolates. Lanes 1: molecular marker; Lane 2: ATCC 14028 Salmonella typhi; Lanes 3 to 9: nosocomial Salmonella Enteritidis isolates; Lane 10: community isolate from the city of Sa ˜o Paulo; Lane 11: community isolate from another city; Lane 12: isolate from the enteral diet.
Discussion Nosocomial outbreaks of Salmonella spp. infection are not uncommon and most hospital outbreaks are small and last days or weeks.3,18 Predisposing host risk factors may facilitate infection by smaller inocula of Salmonella spp., such as the prior use of antibiotics, antacids or H2 antagonists, presence of malignancy, liver disease, haemoglobinopathies, gastrectomy or achlorhydria.19,20 The age of the host is also an important determinant of susceptibility to infection. Children aged less than one year and people over 60 years acquire salmonellosis more frequently than other age groups, and the severity of illness is greater.19,21,22 Such patients may become bacteraemic. In our patients, prior antibiotic use, underlying liver disease and malignancy may have contributed to diarrhoea. Three patients died, but death was not related to salmonellosis, according to their medical records. The vehicle of transmission of Salmonella spp. in this outbreak was considered to be the enteral diet. Salmonellosis associated with enteral nutrition, including typhoid organisms, has not been reported previously. After the detection of a sample of enteral diet as being positive for salmonella, the Infection Control Department searched for nosocomial cases of salmonellosis. As a result, eight cases were identified. The patients were hospitalized in different
units and their underlying diseases were diverse. On review of the medical records, it was found that all patients had received enteral nutrition. The Nutrition and Dietary Department was therefore visited and enteral nutrition preparation, storage and handling procedures were reviewed. No problems were found with the employees or with the diet preparation procedures. All cultures of tubs, taps and filtered water were negative. These data suggested that diet was probably contaminated before re-hydration. In our study, all isolates of Salmonella spp. (clinical and diet samples) were indistinguishable and different from the strain from another city (Carapicuiba), using PFGE typing. One isolate detected on admission, in a blood culture sample from a patient unrelated to the outbreak, was genotypically indistinguishable from the nosocomial strains. This patient had not been hospitalized previously, had not received an enteral diet, and had had no contact with the nosocomial cases. We concluded therefore that in this outbreak, the Salmonella spp. in the nosocomial environment originated from a community source connected to the Salmonella serovars found in the city of Sa ˜o Paulo. Tavechio et al.23 showed an increase in S. enteritidis infection in the State of Sa ˜o Paulo, Brazil, associated with the consumption of raw eggs, as indicated by epidemiological investigation
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of outbreaks. In the late 1970s, strains of S. typhimurium were mainly associated with nosocomial infections in Sa ˜o Paulo.23 – 25 The diet powder contained lyophilized albumin extracted from eggs that could have been the source of salmonella infection. Vaccination of chickens may make egg production safer,26 but in Brazil vaccination of flocks is minimal. There are no regulations, and public awareness about the problem and pressure for control is non-existent. Lyophilization or freeze-drying is a method used for food conservation in which water is removed by sublimation. The material is frozen at 2 40 8F then is heated to very high temperatures. As a result, humidity contained in the food goes directly from the solid to the gaseous state. The procedures and reasons for using lyophilization are varied.27 The effects of lyophilization on the viability and storage of attenuated live microbial vaccines were studied for Salmonella, Shigella and Escherichia coli, and lyophilization was found to maintain the biological, antigenic and genetic characteristics of the fresh microorganisms.28 Bergmann et al.29 showed that lyophilization does not destroy cells. Electron microscopy showed that the process only decreased diameter and caused the cell wall to become wavy, whilst the basic structures of cells remained intact. Sinskey et al.30 showed that freezing at 2 40 8 F did not decrease the viable population of S. typhimurium below that of the initial population. Simon et al.31 did not observe any alteration in virulence of S. typhimurium after lyophilization, although organisms metabolically injured by freezing may have slower growth rates. All these data suggest that the egg albumin in the enteral diets may have been the cause of the outbreak and that lyophilization can be used for food preservation, but to assure sterilization, other methods should be employed.
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