Molecular Evidence that Nasal Carriage of Staphylococcus aureus Plays a Role in Respiratory Tract Infections of Critically Ill Patients
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微生物临床杂志 2005年第7期
Service de Reanimation Medicale, Hpital Gui de Chauliac
Laboratoire de Genetique et Evolution des Maladies Infectieuses (GEMI), Institut de Recherche pour le Developpement (IRD)
Laboratoire de Bacteriologie, Hpital Arnaud de Villeneuve, Montpellier, France
ABSTRACT
The relationship between nasal Staphylococcus aureus carriage and lower respiratory tract infections was studied in 16 critically ill patients. S. aureus strains from nasal and bronchial samples were characterized by pulsed-field gel electrophoresis. In all but one case, nasal and bronchial strains were genetically identical in the same patients.
TEXT
Staphylococcus aureus is a major cause of severe community-acquired and nosocomial pneumonia in intensive care units (ICU) (13). The anterior nares are the main reservoir of S. aureus, and nasal S. aureus carriage appears to play a key role in the pathogenesis of S. aureus infection (5, 7, 11, 19). The aim of this study was to explore if nasal S. aureus carriage is the source of lower respiratory tract infections in critically ill patients.
During the study period (4 November 2000 to 4 January 2002), 402 patients were hospitalized in the medical ICU and 49 had S. aureus lower respiratory tract infections, 28 with community-acquired infections and 21 with nosocomial infections defined by the onset of the first clinical manifestations 72 h after hospital admission. For each patient, S. aureus nasal carriage was investigated by nasal swab on admission and weekly. Pneumonia was diagnosed on clinical, biological, and radiologic criteria and the presence of a colony count of 104 CFU/ml from culture of bronchoalveolar fluid (BAL) or 107 CFU/ml from culture of a tracheobronchial aspirate (TBA) sample. Under these thresholds, a diagnosis of distal bronchitis was considered.
Of the 27 patients with both S. aureus nasal carriage and pulmonary infection, 16 patients (11 men, 5 women; mean age, 51 ± 17 years; simplified acute physiology score II, 51 ± 13 [9]; 15 under mechanical ventilation by the oropharyngeal route) were studied and 32 isolates were taken, 16 from the anterior nares and 16 from bronchial samples. Standard microbiological methods were used for identification and antimicrobial susceptibility testing according to the national recommendations (15). Methicillin resistance was confirmed by the presence of the mecA gene, determined by PCR (12). Molecular analysis using pulsed-field gel electrophoresis (PFGE) was performed on the 32 isolates. DNAs were digested with SmaI (New England Biolabs, Hertfordshire, United Kingdom), and PFGE was performed with a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA).
Methicillin-susceptible S. aureus (MSSA) strains were isolated in both nares and bronchial samples of eight patients and methicillin-resistant S. aureus (MRSA) in the other eight patients. No patient had simultaneous MSSA and MRSA. MSSA nasal carriage was detected on admission in seven patients. MRSA nasal carriage was acquired in the ICU in six and imported in two patients (Table 1). Pneumonia and distal bronchitis were community acquired in seven patients and nosocomial in nine. In 10 cases, S. aureus was isolated in nasal and bronchial samples simultaneously. In five patients, S. aureus was isolated first in nasal samples and secondarily in bronchial samples. In patient 10, S. aureus was recovered in nasal samples 18 days after pneumonia (Table 1).
Antibiotic susceptibility profiles of isolates recovered from both nares and the respiratory tract of the same patient were identical for the 17 antibiotics used in 12 patients and differed by no more than two agents for the other 4 patients (Table 1).
PFGE profiles are shown in Fig. 1. In 15 cases, S. aureus strains obtained from nasal and bronchial samples of the same patients were indistinguishable (Table 1). In the remaining patient (patient 1), the MSSA strains isolated from the nares and bronchial sample differed from each other by 17 DNA fragments in PFGE and displayed different antibiotypes.
Several studies focused on the link between S. aureus nasal carriage, oropharyngeal and tracheal colonization, and pneumonia, but the majority used phenotypic markers such as serotyping or antibiotic susceptibility testing (1, 3, 4, 8, 18). As shown in this study, antibiotyping is often inadequate for differentiating strains. Antibiotic susceptibility patterns were different between strains isolated from nares and strains isolated from the respiratory tract in three patients while these strains were genetically identical using molecular tools. These differences could be explained by acquisition of plasmids or mobile genetic elements carrying antibiotic resistance genes or differences in gene expression between identical genotypes (10).
To our knowledge, only four studies using molecular markers focused on relatedness between S. aureus strains isolated in nasal or oropharyngeal or gastric and bronchial samples in noncritically or critically ill patients (2, 6, 16, 20). All four of them used the PFGE method, but the designs of these studies were different. In our study, we assessed a larger sample of critically ill patients who had nasal carriage and community-acquired or nosocomial respiratory tract infections with MRSA or MSSA. Our PFGE results showed that the S. aureus strain isolated from nares was genetically identical to that isolated from the bronchial sample of the same patient in 15 out of 16 cases. This genetic identity demonstrates a link between S. aureus nasal carriage and S. aureus pneumonia or bronchitis in the majority of critically ill patients. In only one patient were MSSA strains isolated simultaneously in bronchial and nasal swab samples genetically different according to the criteria of Tenover et al. (more than seven different DNA fragments) (17). In studies by Garrouste-Orgeas et al. and Watanabe et al., MRSA strains isolated from oropharyngeal or nasal and bronchial samples were unrelated in one and three cases, respectively (6, 20). Thus, in some cases, nasal carriage does not imply a pulmonary infection by the same strain and nasal and bronchial strains can present different phenotypic characteristics, especially antibiotic susceptibility patterns. Several hypotheses could explain the differences between nasal and bronchial samples: (i) a minority of patients can harbor several different strains in different parts of the body (14); (ii) some strains might have been underdetected because only one colony was selected for PFGE analysis; or (iii) one different strain might have been inoculated by health care workers during tracheal aspiration.
These results are important with regard to understanding the route of S. aureus pneumonia in critically ill patients. Most of the S. aureus strains from pneumonia and bronchitis are derived from the nasal cavity. Oropharyngeal secretions are probably contaminated by S. aureus strains from the nasal cavity, and patients aspirate oropharyngeal secretions when consciousness is altered or during intubation and mechanical ventilation. The severe consequences of S. aureus pneumonia heighten the importance of prevention. Thus, strategies that can eliminate S. aureus nasal carriage may prevent staphylococcal pneumonia.
REFERENCES
Bergmans, D., M. Bonten, C. Gaillard, P. de Leeuw, F. van Tiel, E. Stobberingh, and S. van der Geest. 1996. Clinical spectrum of ventilator-associated pneumonia caused by methicillin-sensitive Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 15:437-445.
Campbell, W., E. Hendrix, R. Schwalbe, A. Fattom, and R. Edelman. 1999. Head-injured patients who are nasal carriers of Staphylococcus aureus are at high risk for Staphylococcus aureus pneumonia. Crit. Care Med. 27:798-801.
Cardenosa Cendrero, J. A., J. Sole-Violan, A. Bordes Benitez, J. Noguera Catalan, J. Arroyo Fernandez, P. Saavedra Santana, and F. Rodriguez de Castro. 1999. Role of different routes of tracheal colonization in the development of pneumonia in patients receiving mechanical ventilation. Chest 116:462-470.
Ewig, S., A. Torres, M. El-Ebiary, N. Fabregas, C. Hernandez, J. Gonzalez, J. M. Nicolas, and L. Soto. 1999. Bacterial colonization patterns in mechanically ventilated patients with traumatic and medical head injury. Incidence, risk factors, and association with ventilator-associated pneumonia. Am. J. Respir. Crit. Care Med. 159:188-198.
Fukuda, M., H. Tanaka, Y. Kajiwara, T. Sugimura, E. Oda, H. Suenaga, M. Yoshimura, T. Iino, M. Togawa, Y. Hirakata, H. Soda, M. Oka, S. Kohno, and T. Oshibuchi. 2004. High-risk populations for nasal carriage of methicillin-resistant Staphylococcus aureus. J. Infect. Chemother. 10:189-191.
Garrouste-Orgeas, M., S. Chevret, G. Arlet, O. Marie, M. Rouveau, N. Popoff, and B. Schlemmer. 1997. Oropharyngeal or gastric colonization and nosocomial pneumonia in adult intensive care unit patients. A prospective study based on genomic DNA analysis. Am. J. Respir. Crit. Care Med. 156:1647-1655.
Kluytmans, J., A. van Belkum, and H. Verbrugh. 1997. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10:505-520.
Kropec, A., J. Huebner, M. Riffel, U. Bayer, A. Benzing, K. Geiger, and F. D. Daschner. 1993. Exogenous or endogenous reservoirs of nosocomial Pseudomonas aeruginosa and Staphylococcus aureus infections in a surgical intensive care unit. Intensive Care Med. 19:161-165.
Le Gall, J. R., S. Lemeshow, and F. Saulnier. 1993. A new simplified acute physiology score (SAPS II) based on a European/North American multicenter study. JAMA 270:2957-2963.
Maslow, J. N., M. E. Mulligan, and R. D. Arbeit. 1993. Molecular epidemiology: application of contemporary techniques to the typing of microorganisms. Clin. Infect. Dis. 17:153-162.
Merrer, J., G. Pisica-Donose, M. Leneveu, and F. Pauthier. 2004. Prevalence of methicillin-resistant Staphylococcus aureus nasal carriage among patients with femoral neck fractures: implication for antibiotic prophylaxis. Infect. Control Hosp. Epidemiol. 25:515-517.
Predari, S. C., M. Ligozzi, and R. Fontana. 1991. Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction. Antimicrob. Agents Chemother. 35:2568-2573.
Rello, J., and E. Diaz. 2003. Pneumonia in the intensive care unit. Crit. Care Med. 31:2544-2551.
Shiseki, M., H. Hidai, K. Kikuchi, and K. Totsuka. 2000. Genotypic similarities among the methicillin-resistant Staphylococcus aureus isolates recovered from stools, the respiratory tract and other sites in 20 patients. J. Hosp. Infect. 45:244-248.
Soussy, C. J., G. Carret, J. D. Cavallo, H. Chardon, C. Chidiac, P. Choutet, P. Courvalin, H. Dabernat, H. Drugeon, L. Dubreuil, F. Goldstein, V. Jarlier, R. Leclercq, M. H. Nicolas-Chanoine, A. Philippon, C. Quentin, B. Rouveix, and J. Sirot. 2000. Antibiogram Committee of the French Microbiology Society. Report 2000-2001. Pathol. Biol. 48:832-871.
Talon, D., C. Rouget, V. Cailleaux, P. Bailly, M. Thouverez, F. Barale, and Y. Michel-Briand. 1995. Nasal carriage of Staphylococcus aureus and cross-contamination in a surgical intensive care unit: efficacy of mupirocin ointment. J. Hosp. Infect. 30:39-49.
Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.
Torres, A., M. el-Ebiary, J. Gonzalez, M. Ferrer, J. Puig de la Bellacasa, A. Gene, A. Martos, and R. Rodriguez-Roisin. 1993. Gastric and pharyngeal flora in nosocomial pneumonia acquired during mechanical ventilation. Am. Rev. Respir. Dis. 148:352-357.
von Eiff, C., K. Becker, K. Machka, H. Stammer, and G. Peters. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. N. Engl. J. Med. 344:11-16.
Watanabe, H., H. Masaki, N. Asoh, K. Watanabe, K. Oishi, S. Kobayashi, A. Sato, and T. Nagatake. 2000. Molecular analysis of methicillin-resistant Staphylococcus aureus as a causative agent of bronchopulmonary infection: relation to colonization in the upper respiratory tract. J. Clin. Microbiol. 38:3867-3869.(Philippe Corne, Helene Ma)
Laboratoire de Genetique et Evolution des Maladies Infectieuses (GEMI), Institut de Recherche pour le Developpement (IRD)
Laboratoire de Bacteriologie, Hpital Arnaud de Villeneuve, Montpellier, France
ABSTRACT
The relationship between nasal Staphylococcus aureus carriage and lower respiratory tract infections was studied in 16 critically ill patients. S. aureus strains from nasal and bronchial samples were characterized by pulsed-field gel electrophoresis. In all but one case, nasal and bronchial strains were genetically identical in the same patients.
TEXT
Staphylococcus aureus is a major cause of severe community-acquired and nosocomial pneumonia in intensive care units (ICU) (13). The anterior nares are the main reservoir of S. aureus, and nasal S. aureus carriage appears to play a key role in the pathogenesis of S. aureus infection (5, 7, 11, 19). The aim of this study was to explore if nasal S. aureus carriage is the source of lower respiratory tract infections in critically ill patients.
During the study period (4 November 2000 to 4 January 2002), 402 patients were hospitalized in the medical ICU and 49 had S. aureus lower respiratory tract infections, 28 with community-acquired infections and 21 with nosocomial infections defined by the onset of the first clinical manifestations 72 h after hospital admission. For each patient, S. aureus nasal carriage was investigated by nasal swab on admission and weekly. Pneumonia was diagnosed on clinical, biological, and radiologic criteria and the presence of a colony count of 104 CFU/ml from culture of bronchoalveolar fluid (BAL) or 107 CFU/ml from culture of a tracheobronchial aspirate (TBA) sample. Under these thresholds, a diagnosis of distal bronchitis was considered.
Of the 27 patients with both S. aureus nasal carriage and pulmonary infection, 16 patients (11 men, 5 women; mean age, 51 ± 17 years; simplified acute physiology score II, 51 ± 13 [9]; 15 under mechanical ventilation by the oropharyngeal route) were studied and 32 isolates were taken, 16 from the anterior nares and 16 from bronchial samples. Standard microbiological methods were used for identification and antimicrobial susceptibility testing according to the national recommendations (15). Methicillin resistance was confirmed by the presence of the mecA gene, determined by PCR (12). Molecular analysis using pulsed-field gel electrophoresis (PFGE) was performed on the 32 isolates. DNAs were digested with SmaI (New England Biolabs, Hertfordshire, United Kingdom), and PFGE was performed with a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA).
Methicillin-susceptible S. aureus (MSSA) strains were isolated in both nares and bronchial samples of eight patients and methicillin-resistant S. aureus (MRSA) in the other eight patients. No patient had simultaneous MSSA and MRSA. MSSA nasal carriage was detected on admission in seven patients. MRSA nasal carriage was acquired in the ICU in six and imported in two patients (Table 1). Pneumonia and distal bronchitis were community acquired in seven patients and nosocomial in nine. In 10 cases, S. aureus was isolated in nasal and bronchial samples simultaneously. In five patients, S. aureus was isolated first in nasal samples and secondarily in bronchial samples. In patient 10, S. aureus was recovered in nasal samples 18 days after pneumonia (Table 1).
Antibiotic susceptibility profiles of isolates recovered from both nares and the respiratory tract of the same patient were identical for the 17 antibiotics used in 12 patients and differed by no more than two agents for the other 4 patients (Table 1).
PFGE profiles are shown in Fig. 1. In 15 cases, S. aureus strains obtained from nasal and bronchial samples of the same patients were indistinguishable (Table 1). In the remaining patient (patient 1), the MSSA strains isolated from the nares and bronchial sample differed from each other by 17 DNA fragments in PFGE and displayed different antibiotypes.
Several studies focused on the link between S. aureus nasal carriage, oropharyngeal and tracheal colonization, and pneumonia, but the majority used phenotypic markers such as serotyping or antibiotic susceptibility testing (1, 3, 4, 8, 18). As shown in this study, antibiotyping is often inadequate for differentiating strains. Antibiotic susceptibility patterns were different between strains isolated from nares and strains isolated from the respiratory tract in three patients while these strains were genetically identical using molecular tools. These differences could be explained by acquisition of plasmids or mobile genetic elements carrying antibiotic resistance genes or differences in gene expression between identical genotypes (10).
To our knowledge, only four studies using molecular markers focused on relatedness between S. aureus strains isolated in nasal or oropharyngeal or gastric and bronchial samples in noncritically or critically ill patients (2, 6, 16, 20). All four of them used the PFGE method, but the designs of these studies were different. In our study, we assessed a larger sample of critically ill patients who had nasal carriage and community-acquired or nosocomial respiratory tract infections with MRSA or MSSA. Our PFGE results showed that the S. aureus strain isolated from nares was genetically identical to that isolated from the bronchial sample of the same patient in 15 out of 16 cases. This genetic identity demonstrates a link between S. aureus nasal carriage and S. aureus pneumonia or bronchitis in the majority of critically ill patients. In only one patient were MSSA strains isolated simultaneously in bronchial and nasal swab samples genetically different according to the criteria of Tenover et al. (more than seven different DNA fragments) (17). In studies by Garrouste-Orgeas et al. and Watanabe et al., MRSA strains isolated from oropharyngeal or nasal and bronchial samples were unrelated in one and three cases, respectively (6, 20). Thus, in some cases, nasal carriage does not imply a pulmonary infection by the same strain and nasal and bronchial strains can present different phenotypic characteristics, especially antibiotic susceptibility patterns. Several hypotheses could explain the differences between nasal and bronchial samples: (i) a minority of patients can harbor several different strains in different parts of the body (14); (ii) some strains might have been underdetected because only one colony was selected for PFGE analysis; or (iii) one different strain might have been inoculated by health care workers during tracheal aspiration.
These results are important with regard to understanding the route of S. aureus pneumonia in critically ill patients. Most of the S. aureus strains from pneumonia and bronchitis are derived from the nasal cavity. Oropharyngeal secretions are probably contaminated by S. aureus strains from the nasal cavity, and patients aspirate oropharyngeal secretions when consciousness is altered or during intubation and mechanical ventilation. The severe consequences of S. aureus pneumonia heighten the importance of prevention. Thus, strategies that can eliminate S. aureus nasal carriage may prevent staphylococcal pneumonia.
REFERENCES
Bergmans, D., M. Bonten, C. Gaillard, P. de Leeuw, F. van Tiel, E. Stobberingh, and S. van der Geest. 1996. Clinical spectrum of ventilator-associated pneumonia caused by methicillin-sensitive Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 15:437-445.
Campbell, W., E. Hendrix, R. Schwalbe, A. Fattom, and R. Edelman. 1999. Head-injured patients who are nasal carriers of Staphylococcus aureus are at high risk for Staphylococcus aureus pneumonia. Crit. Care Med. 27:798-801.
Cardenosa Cendrero, J. A., J. Sole-Violan, A. Bordes Benitez, J. Noguera Catalan, J. Arroyo Fernandez, P. Saavedra Santana, and F. Rodriguez de Castro. 1999. Role of different routes of tracheal colonization in the development of pneumonia in patients receiving mechanical ventilation. Chest 116:462-470.
Ewig, S., A. Torres, M. El-Ebiary, N. Fabregas, C. Hernandez, J. Gonzalez, J. M. Nicolas, and L. Soto. 1999. Bacterial colonization patterns in mechanically ventilated patients with traumatic and medical head injury. Incidence, risk factors, and association with ventilator-associated pneumonia. Am. J. Respir. Crit. Care Med. 159:188-198.
Fukuda, M., H. Tanaka, Y. Kajiwara, T. Sugimura, E. Oda, H. Suenaga, M. Yoshimura, T. Iino, M. Togawa, Y. Hirakata, H. Soda, M. Oka, S. Kohno, and T. Oshibuchi. 2004. High-risk populations for nasal carriage of methicillin-resistant Staphylococcus aureus. J. Infect. Chemother. 10:189-191.
Garrouste-Orgeas, M., S. Chevret, G. Arlet, O. Marie, M. Rouveau, N. Popoff, and B. Schlemmer. 1997. Oropharyngeal or gastric colonization and nosocomial pneumonia in adult intensive care unit patients. A prospective study based on genomic DNA analysis. Am. J. Respir. Crit. Care Med. 156:1647-1655.
Kluytmans, J., A. van Belkum, and H. Verbrugh. 1997. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10:505-520.
Kropec, A., J. Huebner, M. Riffel, U. Bayer, A. Benzing, K. Geiger, and F. D. Daschner. 1993. Exogenous or endogenous reservoirs of nosocomial Pseudomonas aeruginosa and Staphylococcus aureus infections in a surgical intensive care unit. Intensive Care Med. 19:161-165.
Le Gall, J. R., S. Lemeshow, and F. Saulnier. 1993. A new simplified acute physiology score (SAPS II) based on a European/North American multicenter study. JAMA 270:2957-2963.
Maslow, J. N., M. E. Mulligan, and R. D. Arbeit. 1993. Molecular epidemiology: application of contemporary techniques to the typing of microorganisms. Clin. Infect. Dis. 17:153-162.
Merrer, J., G. Pisica-Donose, M. Leneveu, and F. Pauthier. 2004. Prevalence of methicillin-resistant Staphylococcus aureus nasal carriage among patients with femoral neck fractures: implication for antibiotic prophylaxis. Infect. Control Hosp. Epidemiol. 25:515-517.
Predari, S. C., M. Ligozzi, and R. Fontana. 1991. Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction. Antimicrob. Agents Chemother. 35:2568-2573.
Rello, J., and E. Diaz. 2003. Pneumonia in the intensive care unit. Crit. Care Med. 31:2544-2551.
Shiseki, M., H. Hidai, K. Kikuchi, and K. Totsuka. 2000. Genotypic similarities among the methicillin-resistant Staphylococcus aureus isolates recovered from stools, the respiratory tract and other sites in 20 patients. J. Hosp. Infect. 45:244-248.
Soussy, C. J., G. Carret, J. D. Cavallo, H. Chardon, C. Chidiac, P. Choutet, P. Courvalin, H. Dabernat, H. Drugeon, L. Dubreuil, F. Goldstein, V. Jarlier, R. Leclercq, M. H. Nicolas-Chanoine, A. Philippon, C. Quentin, B. Rouveix, and J. Sirot. 2000. Antibiogram Committee of the French Microbiology Society. Report 2000-2001. Pathol. Biol. 48:832-871.
Talon, D., C. Rouget, V. Cailleaux, P. Bailly, M. Thouverez, F. Barale, and Y. Michel-Briand. 1995. Nasal carriage of Staphylococcus aureus and cross-contamination in a surgical intensive care unit: efficacy of mupirocin ointment. J. Hosp. Infect. 30:39-49.
Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.
Torres, A., M. el-Ebiary, J. Gonzalez, M. Ferrer, J. Puig de la Bellacasa, A. Gene, A. Martos, and R. Rodriguez-Roisin. 1993. Gastric and pharyngeal flora in nosocomial pneumonia acquired during mechanical ventilation. Am. Rev. Respir. Dis. 148:352-357.
von Eiff, C., K. Becker, K. Machka, H. Stammer, and G. Peters. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. N. Engl. J. Med. 344:11-16.
Watanabe, H., H. Masaki, N. Asoh, K. Watanabe, K. Oishi, S. Kobayashi, A. Sato, and T. Nagatake. 2000. Molecular analysis of methicillin-resistant Staphylococcus aureus as a causative agent of bronchopulmonary infection: relation to colonization in the upper respiratory tract. J. Clin. Microbiol. 38:3867-3869.(Philippe Corne, Helene Ma)