- Email: [email protected]
PCR based fingerprinting of Enterobacter cloacae
Journal
of Hospital
Infection
(1994)
27, 2X7-240
SHORT
PCR based U. Ni Riain,
REPORT
fingerprinting
M. G. Cormican,
of Enterobacter J. Flynn,
T. Smith
cloacae
and M. Glennon*
Department of Medical Microbiology, University College Hospital, Galway, Ireland and *The National Diagnostics Centre, University College Galway, Ireland, UK Accepted for publication
22 March
1994
Summary:
An outbreak of lower respiratory tract infection with Enterobatter clo&ae occurred in an intensive care -unit in a university teaching hosnital. Random amnlification of nolvmornhic DNA CRAPD> was used to ass&t in the investigation of the outbreak. The techniquk was riadily applied to this organism and permitted differentiation between strains which had identical biochemical profiles and antibiograms. The versatility of this technique makes it attractive for use in hospitals where fingerprinting of any one of the many Gram-negative rods associated with nosocomial infection may be required from time to time. Keywords:
Enterobacter
cloacae;
genotyping;
hospital
outbreak.
Introduction
Enterobacter spp. was first described as a nosocomial pathogen in the early 1980s. Since then, the incidence of nosocomial infection due to E. cloacae has increased significantly’ and it has displayed increasing resistance to antimicrobial agents.’ Most E. cloacae infections are thought to be endogenous, but common source outbreaks and episodes of cross-infection have also been described.’ The typing systems employed in these studies have included biochemical testing, serotyping, phage typing and cloacin typing. More recently, DNAbased typing methods such as restriction fragment length polymorphism (RFLP) analysis of total DNA and ribosomal DNA regions have been described,3 which provide improved discrimination and typability. Random amplification of polymorphic DNA (RAPD) is another DNA method that generates polymorphic DNA fragments by PCR amplification using oligonucleotides of random sequence. The technique has been applied to a variety of species of bacteria, requires less DNA than RFLP typing, and is rapid and convenient.4,5 Recently a small E. cloacae outbreak occurred in the intensive care unit 0195-6701/94/070237+04
$08.00/O
0 1994 The HospA
237
Infectmn
Smety
238
U. Ni Riain et al.
of our hospital. RAPD proved a convenient technique for the definition of the outbreak strain, with discrimination superior to that achieved by the use of biochemical profile and antibiogram. Materials
and methods
Isolation and identification of strains Sputum specimens from the patients were cultured on a number of media, including cysteine lactose electrolyte deficient (CLED) medium. Environmental swabs were cultured on CLED medium to which cefotaxime discs (30 lg) were applied to facilitate identification of cefotaxime-resistant isolates for further analysis. Isolates were identified by the API 20E system and sensitivity testing was performed on diagnostic sensitivity test medium by the modified Stokes’ method. Two strains of E. cloacae from another hospital (kindly donated by Dr Falkiner, St James’ Hospital, Dublin) were included as external controls. DNA extraction E. cloacae isolates were cultured in peptone water at 37°C overnight with gentle shaking. Bacteria were harvested by centrifugation and the pellet resuspended in TE buffer [lo mM Tris-HCL pH 8.0, 1 mM ethylenediamine-N,N’-tetraacetic acid (EDTA)]. Bacteria were lysed by digestion with proteinase K (1 mg ml-‘) in sodium dodecyl sulphate (1%). The DNA was extracted as described by van Soolingen et aZ.6 PCR and RAPD fingerprinting PCR was performed in 25 ~1 volumes containing 20 ng of template DNA, 3 mM MgC12, 800 nM primer, 1 U Taq polymerase (Promega), 250 FM each of dATP, dCTP, dTTP and dGTP, 10 mM Tris-HCl (pH S.O), 50 mM KC1 and 0.1% Triton x 100 and 0.01% gelatin. The reaction was performed in a Perkin-Elmer cycler under 1 drop of mineral oil. The sequence of the primer used was 5’-AGCAGCGTGG-3’. The cycling program was as follows. Four cycles of 94°C for 5 min, 36°C for 5 min, 72°C for 5 min; followed by 30 cycles of 94°C for 1 min, 36°C for 1 min, 72°C for 1 min; and then 72°C for 10 min. A 20 ~1 sample of the reaction volume was loaded onto a 15% agarose gel containing 0.5 ng ml-’ of ethidium bromide for electrophoresis and the banding patterns were visualized by U.V. illumination. Results Three clinical isolates of E. cloacae (A, B and C) from patients nursed simultaneously in the intensive care unit, all exhibiting resistance to the extended spectrum cephalosporins, alerted us to the possibility of a single
1
2
3
4
239
and E. cloacae
PCR 5
6
7
8
9
10
11
Figure 1. Random amplification of polymorphic DNA patterns generated from E. cloacae DNA following electrophoresis and ethidium bromide staining. Lanes 1, 2 and 3, isolate A showing reproducibility of pattern between PCR runs (lanes 1 and 2) and within a PCR run (lanes 2 and 3); lanes 4 and 5, isolates B and C; lanes 6 and 7, isolates D and E; lanes 8 and 9, isolates F and G; lanes 10 and 11, external isolates.
strain outbreak. The isolates with the API 20E system. All
produced identical biochemical profiles were resistant to ampicillin, cephradine,
cefuroxime, cefotaxime and azlocillin, intermediately resistant to piperacillin, and sensitive to gentamicin, amikacin and ofloxacin. Extensive environmental sampling identified four other E. cloacae isolates (D, E, F and G) that were resistant to cefotaxime. Of these, two isolates (D and E) from equipment used in the care of the index patient (the ventilator trap and a suction jug) and one isolate (F) from the floor adjacent to the bed station of the index patient, had identical biochemical profiles and antibiograms to the clinical isolates. The fourth environmental isolate (G), which was also made from the floor adjacent to the index patient’s bed, had a slightly different antibiogram (intermediate resistance to cefotaxime). RAPD fingerprinting confirmed the identity of the three clinical isolates and the two isolates from equipment used in the care of the index patient (Figure 1). The isolates from the floor adjacent to the bedside and two isolates from another hospital, were clearly different. The banding patterns
240
U. Ni Riain
et al.
were consistent for the same isolate both within a given PCR run and in different runs (Figure l), thus demonstrating reproducibility. Discussion
The importance of a high standard of infection control practice in preventing cross-infection with antibiotic-resistant organisms is well recognized. In this outbreak a review of infection control practice and isolation of the index patient resulted in termination of the outbreak. As a result of the outbreak and the environmental sampling, we had a group of six E. cloacae isolates that were phenotypically identical, and one isolate similar to this group. Of the six identical isolates, one was isolated from the floor adjacent to the index patient’s bed, suggesting contamination of the general environment. It was considered that this might be related to contamination during emptying of the condensate in the ventilator trap. We studied these isolates in more detail to determine if the phenotypic similarities reflected a close relationship at the DNA level. RAPD was applied to this group of isolates of E. cloacae and yielded reproducible and interpretable results. We were able to demonstrate that the isolate from the general environment which was phenotypically related to the outbreak strain differed at a genomic level, and to confirm the identity of the remaining five isolates. In order to fingerprint the diverse Gram-negative bacilli which are associated with nosocomial outbreaks of infections, a simple technique with broad applicability is needed, particularly for typing small groups of isolates. We believe that the simplicity of RAPD fingerprinting and its applicability to a wide variety of organisms may satisfy this requirement. We wish to acknowledge the provided us with the external photographic assistance.
assistance of Dr Falkiner, isolates of E. cZoacae, and
St James’ are grateful
Hospital, to Stuart
Dublin, Baker
for
who his
References
;: 3. 4. 5. 6.
Falkiner FR. Enterobacter in hospital. r Hasp Infect 1992; 20: 137-140. John JF, Sharbaugh RJ, Bannister ER. Enterobacter cloacae bacteraemia, epidemiology, and antibiotic resistance. Rev Infect Dis 1982; 4: 13-28. Bingen E, Denamur E, Lambert-Zechovsky N, Brahimi N, El Lakany M, Elion J. Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections. J Hasp Infect 1992; 21: 95-101. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992; 20: 5137-5142. Matthews RC. PCR fingerprinting microbes by random amplification of polymorphic DNA. J Med Microbial 1992; 37: 286-290. van Soolingen D, Hermans PWM, de Haas PEW, Sol1 DR, van Embden JDA. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: Evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbial 1991; 29: 2578-2586.
of Hospital
Infection
(1994)
27, 2X7-240
SHORT
PCR based U. Ni Riain,
REPORT
fingerprinting
M. G. Cormican,
of Enterobacter J. Flynn,
T. Smith
cloacae
and M. Glennon*
Department of Medical Microbiology, University College Hospital, Galway, Ireland and *The National Diagnostics Centre, University College Galway, Ireland, UK Accepted for publication
22 March
1994
Summary:
An outbreak of lower respiratory tract infection with Enterobatter clo&ae occurred in an intensive care -unit in a university teaching hosnital. Random amnlification of nolvmornhic DNA CRAPD> was used to ass&t in the investigation of the outbreak. The techniquk was riadily applied to this organism and permitted differentiation between strains which had identical biochemical profiles and antibiograms. The versatility of this technique makes it attractive for use in hospitals where fingerprinting of any one of the many Gram-negative rods associated with nosocomial infection may be required from time to time. Keywords:
Enterobacter
cloacae;
genotyping;
hospital
outbreak.
Introduction
Enterobacter spp. was first described as a nosocomial pathogen in the early 1980s. Since then, the incidence of nosocomial infection due to E. cloacae has increased significantly’ and it has displayed increasing resistance to antimicrobial agents.’ Most E. cloacae infections are thought to be endogenous, but common source outbreaks and episodes of cross-infection have also been described.’ The typing systems employed in these studies have included biochemical testing, serotyping, phage typing and cloacin typing. More recently, DNAbased typing methods such as restriction fragment length polymorphism (RFLP) analysis of total DNA and ribosomal DNA regions have been described,3 which provide improved discrimination and typability. Random amplification of polymorphic DNA (RAPD) is another DNA method that generates polymorphic DNA fragments by PCR amplification using oligonucleotides of random sequence. The technique has been applied to a variety of species of bacteria, requires less DNA than RFLP typing, and is rapid and convenient.4,5 Recently a small E. cloacae outbreak occurred in the intensive care unit 0195-6701/94/070237+04
$08.00/O
0 1994 The HospA
237
Infectmn
Smety
238
U. Ni Riain et al.
of our hospital. RAPD proved a convenient technique for the definition of the outbreak strain, with discrimination superior to that achieved by the use of biochemical profile and antibiogram. Materials
and methods
Isolation and identification of strains Sputum specimens from the patients were cultured on a number of media, including cysteine lactose electrolyte deficient (CLED) medium. Environmental swabs were cultured on CLED medium to which cefotaxime discs (30 lg) were applied to facilitate identification of cefotaxime-resistant isolates for further analysis. Isolates were identified by the API 20E system and sensitivity testing was performed on diagnostic sensitivity test medium by the modified Stokes’ method. Two strains of E. cloacae from another hospital (kindly donated by Dr Falkiner, St James’ Hospital, Dublin) were included as external controls. DNA extraction E. cloacae isolates were cultured in peptone water at 37°C overnight with gentle shaking. Bacteria were harvested by centrifugation and the pellet resuspended in TE buffer [lo mM Tris-HCL pH 8.0, 1 mM ethylenediamine-N,N’-tetraacetic acid (EDTA)]. Bacteria were lysed by digestion with proteinase K (1 mg ml-‘) in sodium dodecyl sulphate (1%). The DNA was extracted as described by van Soolingen et aZ.6 PCR and RAPD fingerprinting PCR was performed in 25 ~1 volumes containing 20 ng of template DNA, 3 mM MgC12, 800 nM primer, 1 U Taq polymerase (Promega), 250 FM each of dATP, dCTP, dTTP and dGTP, 10 mM Tris-HCl (pH S.O), 50 mM KC1 and 0.1% Triton x 100 and 0.01% gelatin. The reaction was performed in a Perkin-Elmer cycler under 1 drop of mineral oil. The sequence of the primer used was 5’-AGCAGCGTGG-3’. The cycling program was as follows. Four cycles of 94°C for 5 min, 36°C for 5 min, 72°C for 5 min; followed by 30 cycles of 94°C for 1 min, 36°C for 1 min, 72°C for 1 min; and then 72°C for 10 min. A 20 ~1 sample of the reaction volume was loaded onto a 15% agarose gel containing 0.5 ng ml-’ of ethidium bromide for electrophoresis and the banding patterns were visualized by U.V. illumination. Results Three clinical isolates of E. cloacae (A, B and C) from patients nursed simultaneously in the intensive care unit, all exhibiting resistance to the extended spectrum cephalosporins, alerted us to the possibility of a single
1
2
3
4
239
and E. cloacae
PCR 5
6
7
8
9
10
11
Figure 1. Random amplification of polymorphic DNA patterns generated from E. cloacae DNA following electrophoresis and ethidium bromide staining. Lanes 1, 2 and 3, isolate A showing reproducibility of pattern between PCR runs (lanes 1 and 2) and within a PCR run (lanes 2 and 3); lanes 4 and 5, isolates B and C; lanes 6 and 7, isolates D and E; lanes 8 and 9, isolates F and G; lanes 10 and 11, external isolates.
strain outbreak. The isolates with the API 20E system. All
produced identical biochemical profiles were resistant to ampicillin, cephradine,
cefuroxime, cefotaxime and azlocillin, intermediately resistant to piperacillin, and sensitive to gentamicin, amikacin and ofloxacin. Extensive environmental sampling identified four other E. cloacae isolates (D, E, F and G) that were resistant to cefotaxime. Of these, two isolates (D and E) from equipment used in the care of the index patient (the ventilator trap and a suction jug) and one isolate (F) from the floor adjacent to the bed station of the index patient, had identical biochemical profiles and antibiograms to the clinical isolates. The fourth environmental isolate (G), which was also made from the floor adjacent to the index patient’s bed, had a slightly different antibiogram (intermediate resistance to cefotaxime). RAPD fingerprinting confirmed the identity of the three clinical isolates and the two isolates from equipment used in the care of the index patient (Figure 1). The isolates from the floor adjacent to the bedside and two isolates from another hospital, were clearly different. The banding patterns
240
U. Ni Riain
et al.
were consistent for the same isolate both within a given PCR run and in different runs (Figure l), thus demonstrating reproducibility. Discussion
The importance of a high standard of infection control practice in preventing cross-infection with antibiotic-resistant organisms is well recognized. In this outbreak a review of infection control practice and isolation of the index patient resulted in termination of the outbreak. As a result of the outbreak and the environmental sampling, we had a group of six E. cloacae isolates that were phenotypically identical, and one isolate similar to this group. Of the six identical isolates, one was isolated from the floor adjacent to the index patient’s bed, suggesting contamination of the general environment. It was considered that this might be related to contamination during emptying of the condensate in the ventilator trap. We studied these isolates in more detail to determine if the phenotypic similarities reflected a close relationship at the DNA level. RAPD was applied to this group of isolates of E. cloacae and yielded reproducible and interpretable results. We were able to demonstrate that the isolate from the general environment which was phenotypically related to the outbreak strain differed at a genomic level, and to confirm the identity of the remaining five isolates. In order to fingerprint the diverse Gram-negative bacilli which are associated with nosocomial outbreaks of infections, a simple technique with broad applicability is needed, particularly for typing small groups of isolates. We believe that the simplicity of RAPD fingerprinting and its applicability to a wide variety of organisms may satisfy this requirement. We wish to acknowledge the provided us with the external photographic assistance.
assistance of Dr Falkiner, isolates of E. cZoacae, and
St James’ are grateful
Hospital, to Stuart
Dublin, Baker
for
who his
References
;: 3. 4. 5. 6.
Falkiner FR. Enterobacter in hospital. r Hasp Infect 1992; 20: 137-140. John JF, Sharbaugh RJ, Bannister ER. Enterobacter cloacae bacteraemia, epidemiology, and antibiotic resistance. Rev Infect Dis 1982; 4: 13-28. Bingen E, Denamur E, Lambert-Zechovsky N, Brahimi N, El Lakany M, Elion J. Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections. J Hasp Infect 1992; 21: 95-101. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992; 20: 5137-5142. Matthews RC. PCR fingerprinting microbes by random amplification of polymorphic DNA. J Med Microbial 1992; 37: 286-290. van Soolingen D, Hermans PWM, de Haas PEW, Sol1 DR, van Embden JDA. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: Evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbial 1991; 29: 2578-2586.