Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan

Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum beta-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.     

RAPD typing of Campylobacter jejuni and comparison with Lior's or Penner's serotyping system

Campylobacter jejuni were isolated from 7 epidemic outbreaks (121 isolates), 15 patients with gastroenteritis, chicken meats (47 isolates) and chicken cecal contents (70 isolates). The isolates and one standard strain of C. jejuni JCM2013 were analysed by randomly amplified polymorphic DNA method (RAPD). Total of 254 C. jejuni isolates were divided 68 different RAPD types which included strains that did not to divided by Lior's or Penner's serotyping system. To compare the similarities of RAPD patterns among the isolates, the amplification patterns of DNA were estimated by means of the Dice coefficient, and clustering of strains was based on the unweighted average pair group method (UPGMA) to facilitate the plotting of a dendrogram. It suggests that amplification band patterns of human isolates were different from those of chicken ones. Thus additional information given from RAPD profiles serves for epidemiological investigation and RAPD analysis is recommended as rapid and effective typing method.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Transcontinental importation into the UK of Escherichia coli expressing a plasmid-mediated AmpC-type beta-lactamase exposed during an outbreak of SHV-5 extended-spectrum beta-lactamase in a Leeds hospital

Sixteen strains of Escherichia coli with high-level resistance to extended-spectrum cephalosporins and other classes of antibiotic have been isolated at St James' University Hospital, Leeds. They produce up to three separate beta-lactamases: TEM-1, SHV-5 and, in five isolates, a plasmid-mediated AmpC-type enzyme. With the exception of carbapenems, the isolates reported in this study were resistant to all beta-lactam antibiotics including extended-spectrum cephalosporins and the monobactam aztreonam. There was evidence of the spread of a plasmid encoding SHV-5, particularly amongst patients on the liver transplant unit. Sensitivity to beta-lactam antibiotics in five isolates expressing the AmpC-type beta-lactamase was not restored by the beta-lactamase inhibitor clavulanic acid. These bacteria also carried blaSHV-5 on a large plasmid. PCR-amplification of the structural gene and digestion with restriction endonucleases demonstrated that the plasmid-mediated blaAmpC probably identified as BIL-1 using the criteria available. Four of the five patients carrying isolates that carried the plasmid-located blaAmpC gene had recently visited the Indian subcontinent and we presume that they returned carrying these bacteria. Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis (PFGE) suggests that at least four distinct strains existed amongst these five isolates. The two isolates that had very similar PFGE patterns had different plasmid profiles and were isolated from different locations in the hospital and at different times. This study demonstrates the ease with which highly resistant bacteria can be imported into the UK and spread within hospitals.     

Epidemiological study of an outbreak due to multidrug-resistant Enterobacter aerogenes in a medical intensive care unit

In 1993, 63 isolates of Enterobacter aerogenes were collected from 41 patients in a medical intensive care unit (ICU). During the same period, only 46 isolates from 32 patients were collected in the rest of the hospital. All isolates were analyzed by antibiotic resistance phenotype, and 77 representative isolates were differentiated by plasmid restriction analysis, ribotyping, and arbitrarily primed (AP)-PCR. The extended-spectrum beta-lactamases produced by 22 strains were characterized by determination of their isoelectric points and by hybridization of plasmid DNA with specific probes. The isolates were divided into 25 antibiotic resistance phenotypes, either susceptible (group I) or resistant (group II) to aminoglycosides, and exhibited three phenotypes of resistance to beta-lactams: chromosomally derepressed cephalosporinase alone or associated with either extended-spectrum beta-lactamases (mainly of the SHV-4 type) or imipenem resistance. The results of the tests divided the 77 representative isolates (group I, n = 21; group II, n = 56) into 15 plasmid profiles, 14 ribotypes, and 15 AP-PCR patterns. Although the resistant isolates (group II) exhibited different plasmid profiles, ribotyping and AP-PCR analysis demonstrated an identical chromosomal pattern, indicating an epidemiological relatedness. They were mainly found in the medical ICU and occasionally in other units. The susceptible strains (group I) had various and distinct markers and were mainly isolated in units other than the medical ICU. In conclusion, the presence of a nosocomial outbreak in an ICU and the spread of a multidrug-resistant epidemic strain throughout the hospital was confirmed. Ribotyping and AP-PCR represent discriminatory tools for the investigation of nosocomial outbreaks caused by E. aerogenes.     

Note: characterization of Vibrio cholerae O139 Bengal isolated from water in Malaysia

Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.     

The emerging diversity of the electrophoretic types of Vibrio cholerae in the Western Hemisphere

Since the Latin American cholera epidemic began in 1991, 447 isolates of Vibrio cholerae O1 from the Western Hemisphere have been assayed by multilocus enzyme electrophoresis (MEE) to determine allelic variation among 16 enzyme-encoding genes. Two electrophoretic types (ETs) were identified among toxigenic isolates from Latin America: 323 were ET 4, the ET associated with the Latin American epidemic, and 29 were ET 3. Twenty-three of these ET 3 isolates had a distinctive antimicrobial resistance pattern also seen in isolates imported into the United States from Latin America and Southeast Asia. These resistant isolates had an identical ribotype and nearly identical pulsed-field gel electrophoresis (PFGE) patterns. Most nontoxigenic isolates analyzed were not precursors or descendants of toxigenic epidemic strains. MEE provided a population genetic frame-work for the interpretation of PFGE and ribotype data from the isolates in this study. All three methods identified 2 distinct strains of toxigenic V. cholerae O1 currently epidemic in Latin America.     

Molecular typing of Cryptococcus neoformans: taxonomic and epidemiological aspects

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.     

Value of phenotyping methods as an initial screening of Pseudomonas aeruginosa in epidemiologic studies

When studying the epidemiology of Pseudomonas aeruginosa, determination of the similarity of isolates is crucial. In the present study the distinctive capacity of four phenotyping methods (antibiotic susceptibility patterns, serotyping, phage-typing and outer membrane protein [OMP] profile analysis) was determined and compared to pulsed-field gel electrophoresis (PFGE) of enzyme restricted chromosomal DNA. In all, 91 isolates of P. aeruginosa were cultured from ten patients. Antibiotic susceptibility patterns were concordant for all isolates. Serotyping yielded five, phage-typing eight, OMP profile analysis nine and PFGE seven distinct types of P. aeruginosa. Compared to PFGE, the distinctive capacities were 89% (81/91) for serotyping, 87% (79/91) for phage-typing, and 90% (82/91) for OMP profile analysis. When serotyping results were different, PFGE types also were different (exclusiveness 100%). However, isolates with the same serotype may have various PFGE patterns. In contrast, isolates with similar PFGE patterns could have different phage-types or OMP types. For the study of isolates of P. aeruginosa, serotyping provides a good initial selection to reduce the number of isolates that need to be genotyped.     

Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.     

Antimicrobial susceptibilities and molecular epidemiology of Salmonella enterica serotype enteritidis strains isolated in Hong Kong from 1986 to 1996

The incidence of salmonellosis has been increasing in Hong Kong since 1989. The most common Salmonella enterica serotype isolated in 1994 was S. enteritidis. The antimicrobial susceptibilities and molecular epidemiology of 275 S. enteritidis strains isolated in this locality between 1986 and 1996 were studied. Over 99% of the isolates were susceptible to 17 of the 19 antimicrobial agents tested. One isolate harbored an autotransferring plasmid that confers resistance to tetracycline and trimethoprim-sulfamethoxazole. Another isolate harbored a mobilizable plasmid that confers resistance to ampicillin and cephalothin. This isolate was found to produce a beta-lactamase with a pI of 5.2. A total of 264 isolates (96%) were found to harbor one to five plasmids, and the majority (254) harbored a 60-kb plasmid. Of these isolates, 94% contained identical 60-kb plasmids. Based on plasmid profiles, plasmid and chromosomal fingerprints, ribotypes, and randomly amplified polymorphic DNA (RAPD) patterns, 170 (62%) isolates were allocated to group 1b. About 90% of isolates had identical or similar DNA fingerprints, ribotypes, and RAPD patterns, suggesting that a predominant clone of S. enteritidis was circulating in Hong Kong during the period being studied.     

Evidence of genomic instability in Campylobacter jejuni isolated from poultry

Poultry isolates of Campylobacter jejuni derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with SmaI were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.     

Molecular analysis by pulsed-field gel electrophoresis and antibiogram of Streptococcus pneumoniae serotype 6B isolates from selected areas within the United States

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (+/-1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.     

Gaining control: family members relate to persons with severe mental illness

Nine isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected in a Warsaw hospital in 1996 were typed by phenotypic (resistograms) and genotypic (PFGE and plasmid restriction analysis-REAP) methods. Twenty-four (MRSA) strains collected in this hospital during a period of the same duration in 1992 and typed earlier using resistograms and PFGE were also typed by REAP. Comparison of typing results obtained for isolates from 1992 and 1996 showed that strains characterised by PFGE patterns of two distinct types described as specific of the two clonally related groups of Polish MRSA in a multicentre study in 1992 are continuously present in the hospital. However, MRSA strains representing PFGE patterns not observed before were also found within the collection from 1996. REAP typing has proved to have a discriminatory power similar to that of PFGE analysis. Nevertheless, due to the lack of plasmids or difficulties in plasmid DNA isolation in 3 out of 33 studied strains, the typability of REAP turned out to be lower than that of PFGE.     

Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis

We examined genetic variation among 78 clinical isolates of Bordetella pertussis, including 54 strains recovered during a 1986 pertussis epidemic. A total of 16 pulsed-field gel electrophoresis (PFGE) profiles, generated with each of three different enzymes (XbaI, SpeI, and DraI), were obtained from the epidemic and sporadic isolates included in the study. Indistinguishable profiles were seen among strains unrelated temporally or geographically, as well as among strains isolated sporadically from the same geographic areas. All isolates from the epidemic had indistinguishable PFGE profiles. The PFGE pattern of the epidemic strains was shared with only 1 of 25 strains isolated independently of the outbreak. This isolate was cultured from a specimen from a laboratory scientist who had been working with the epidemic strains, further implicating the usefulness of PFGE for the epidemiologic study of clinical strains of B. pertussis. Differences in PFGE profiles for single epidemic strains occurred occasionally upon repeated passage on agar medium, suggesting that subculturing of initial isolates should be minimized before pulsed-field analysis.     

Insertion possibility of 16S-23S space amplification and random amplified polymorphic DNA analysis for typing of methicillin-resistant Staphylococcus aureus strains in the context of nosocomial infections

Within the scope of the present study n = 183 MRSA isolates from the extended area of Düsseldorf and n = 93 international MRSA strains from seven different countries were typed by pulsed-field gel electrophoresis and two PCR methods (RAPD and 16S-23S-spacer amplification). The isolates could be subdivided into 30 different types by PFGE, into 21 by means of RAPD and 18 by 16S-23S-spacer amplification. PFGE had the highest discriminatory potential, however, a combined use of the three typing methods allows a more detailed differentiation even of those isolates with identical PFGE pattern. Both amplification procedures were rapid, easy in handling with reproductable results. For a temporary epidemiological analysis within 24 hours, both amplification methods could be combined. In case the investigated isolates were still suspected of showing a "clonal identity", they should be analysed by additional PFGE (lasting about four days). Although the international isolates were chosen by random selection, several MRSA strains with identical pattern could be found in different countries of the world. Some RAPD-, spacer- and PFGE pattern were constant over many years. This reflects a high genetic stability of single strains.     

Shigellae isolated in 1997--plasmid profiles and antibiotic resistance

INTRODUCTION: Shigella spp is one of the most frequently isolated bacteria causing acute diarrhea with us. Genetics of pathogenicity of Shigella spp. includes chromosomal and plasmid genes. Most virulence factors are coded by invasion plasmid antigen genes residing on a 180-230 MDa plasmid. There is a big problem with multiple resistance of Shigella spp. strains, which is mostly plasmid-borne. Genetic analysis of bacterial cells, that is plasmid profile analysis, is important for investigation of sources and ways of spreading of the infection. All isolates originating from the same clone have identical plasmid profiles, i.e. number and size of plasmids. The aim of the investigation was: comparing the type of resistance to antimicrobical agents found in epidemic and nonepidemic. Shigella strains isolated in 1997, analyzing plasmid profiles of these isolates and confirming their epidemic connection. MATERIAL AND METHODS: Susceptibility to antibiotics was examined by a standard disc-diffusion method. Plasmid profiles of 40 strains (20 from the outbreak and 20 from sporadic cases) were tested using a method of alkaline lysis by Birnboim and Doly followed by electrophoresis in agarose gel. RESULTS: Shigella strains were resistant to antimicrobial agents which are most commonly used. Epidemic isolates shared the same resistance type, they were resistant to cephalexin, streptomycin and co-trimoxazole. The dominant type of resistance of nonepidemic strains was to ampicillin, streptomycin and co-trimoxazole. Strains isolated during the outbreak had identical plasmid profiles (2 plasmid bands of 55 and 1.5 MDa). Non-epidemic isolates had different plasmid profiles as well as type of resistance. CONCLUSION: Strains of Shigella spp. isolated during an outbreak had the same type of resistance and the same plasmid profiles, which indicated their origin from the same clone. The plasmid profile analysis is a reliable and precise method for determination of epidemic connection of Shigella isolates.     

A prolonged outbreak of Shigella sonnei infections in traditionally observant Jewish communities in North America caused by a molecularly distinct bacterial subtype

During 1994-1996, Shigella sonnei outbreaks occurred in 8 North American traditionally observant Jewish communities. These communities remain relatively separate from neighboring populations while maintaining close contact by travel with coreligionists in other cities. Epidemiologic investigations suggested community-to-community transmission via travel. Outbreak-related and control isolates of S. sonnei from each city were subtyped by pulsed-field gel electrophoresis (PFGE) to confirm an epidemiologic linkage between outbreaks. Forty-three (94%) of 46 outbreak-related isolates had closely related PFGE patterns, constituting a single subtype; 33 (94%) of 35 control isolates demonstrated unrelated PFGE patterns. Several patterns differing by < or = 3 bands were identified within the outbreak subtype; one of these accounted for 65% of outbreak isolates. Hence, a single subtype of S. sonnei caused an international outbreak involving 8 traditionally observant Jewish communities, but not neighboring populations, over a 2-year period, suggesting sustained propagation of the epidemic strain between communities.     

Use of capillary electrophoresis in the study of ligand-DNA interactions

The presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains of Salmonella enterica serotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry the aadB, catB3, oxa1, aadA1a, aacA4, and aacC1 gene cassettes. Integrons In-t1 (aadB and catB3) and In-t2 (oxa1 and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4, aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonella strains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the same Salmonella isolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.     

Molecular epidemiological study of nosocomial Enterobacter aerogenes isolates in a Belgian hospital

In 1995, the rate of isolation of Enterobacter aerogenes in the Saint-Pierre University Hospital in Brussels, Belgium, was higher than that in the preceding years. A total of 45 nosocomial E. aerogenes strains were collected from 33 patients of different units during that year, and they were isolated from 19 respiratory specimens, 13 pus specimens, 7 blood specimens, 4 urinary specimens, 1 catheter specimen, and 1 heparin vial. The strains were analyzed to determine their epidemiological relatedness and were characterized by their antibiotic resistance pattern determination, plasmid profiling, and genomic fingerprinting by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). The majority of the strains (82%) were multiply resistant to different commonly used antibiotics. Two major plasmid profiles were found: most strains (64%) harbored two plasmids of different sizes, whereas the others (20%) contained a single plasmid. PFGE with SpeI and/or XbaI restriction enzymes revealed that a single clone (80%) was responsible for causing infections or colonizations throughout the year, and this result was concordant with those obtained by plasmid profiling, with slight variations. By comparing the results of these three methods, PFGE and plasmid profiling were found to be the techniques best suited for investigating the epidemiological relatedness of E. aerogenes strains, and they are therefore proposed as useful tools for the investigation of nosocomial outbreaks caused by this organism.