Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.     

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.     

Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) for typing strains of vancomycin-resistant Enterococcus faecium

In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of a novel assay based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) was tested. A large number of vancomycin-resistant Enterococcus faecium (VREM) isolates from haematological ward patients of the Clinical Hospital in Gdańsk were examined. We found that ADSRRS fingerprinting analysis is a rapid method that offers good discriminatory power. The method demonstrated also excellent reproducibility. The usefulness of the ADSRRS fingerprinting method for molecular typing was compared with pulsed field gel electrophoresis (PFGE) method, which is currently considered the gold standard for molecular typing of isolates recovered from patients and the environment in the course of investigation and control of nosocomial outbreaks. Clustering of ADSRRS fingerprinting data matched pulsed field gel electrophoresis data.The features of ADSRRS fingerprinting technique is discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning VREM that may occur in a single medical ward.     

Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila

Pulsedfield gel electrophoresis (PFGE) was used to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections that occurred over approximately 1 month in a French hospital. Five isolates from patients and 10 isolates from the water supply were characterized by biotyping and antibiotic susceptibility patterns and compared with 10 epidemiologically unrelated strains isolated from patients and rivers, by PFGE of digests of chromosomal DNA. Five environmental and four clinical isolates belonged to the same biotype and antibiotic susceptibility pattern type. The endonucleases XbaI, SpeI and SwaI gave satisfactory profiles whereas DraI did not. The profiles were stable, reproducible and discriminatory. The 10 epidemiologically unrelated strains exhibited 10 different patterns after digestion with XbaI , the least expensive, suitable endonuclease. PFGE is a rapid and discriminatory technique for the typing of Aeromonas hydrophila where a common origin of infection is suspected.     

Genomic heterogeneity of environmental and clinical aeromonads

Thirty-three isolates of Aeromonas from environmental sources and clinical samples were tested and the results, obtained using the pulsed field gel electrophoresis (PFGE) technique, were compared with those obtained by biochemical typing. On the basis of their biochemical characteristics 31 strains was assigned to one of the recognised groups or species within the Aeromonas genus and 2 strains to the species Vibrio fluvialis. These latter were nevertheless found to belong to the Aeromonas genus on the basis of the chromosomal DNA analysis. Among the clinical isolates the biochemical analysis showed greater uniformity. A low correlation between molecular and traditional typing methods was observed with a wider heterogeneity at the genomic level. The results showed the difficulty of discriminating Aeromonas isolates by conventional biochemical methods. The genomic analysis performed by PFGE can be a more effectual technique, which can be used for epidemiological and ecological studies of the microorganisms belonging to the Aeromonas group.     

Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.     

Epidemiologic Typing of Methicillin-Resistant Staphylococcus aureus in Neonate Intensive Care Units Using Pulsed-Field Gel Electrophoresis

To elucidate the mode of dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in neonate intensive care units (NICUs), a total of 223 isolates from 3 separate hospitals were investigated between 1994 and 1996 by a DNA fingerprinting technique with pulsed-field gel electrophoresis (PFGE). Exoprotein profiles of some strains were also examined using SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) and the assessment of enzyme/toxin production such as coagulase, enterotoxin and TSST-1. Judging from the strain typing data from PFGE results and the epidemiological data, 2 different types of PFGE patterns (A and B) and their subtypes (A′, A″ and B′) were identified. The A type including A′ and A″ (comprising approximately 95% of the isolates) was markedly dominant. Only 5% of the isolates belonged to type B and subtype B′. On the other hand, MRSA isolated from adult patients admitted to the same hospital showed many different PFGE patterns. The results strongly suggested that some strain(s) with specific PFGE pattern(s) is prevalent in NICUs. Furthermore, isolates which expressed the same PFGE pattern did not always express the same SDS-PAGE pattern. There were some isolates with different abilities to produce coagulase, enterotoxin C and toxic-shock syndrome toxin (TSST)-1, and the abilities had no relation with a particular type of PFGE pattern. Therefore, a combination of PFGE analysis and biochemical analyses of coagulase, enterotoxin C and TSST-1 may provide us with more detailed information for the epidemiological study of MRSA in NICUs.     

Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.     

Molecular epidemiology of Pseudomonas aeruginosa from cystic fibrosis in Sicily: genome macrorestriction analysis and rapid PCR-ribotyping

This study addresses the epidemiologic relatedness of a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients attending the Pediatric Clinic, Catania, Sicily. Genome macrorestriction analysis after pulsed field gel electrophoresis (PFGE) was used to characterise all strains. Furthermore, a rapid typing procedure, developed in this study, based on polymerase chain reaction amplified ribosomal DNA spacer polymorphisms (PCR-ribotyping), straight from bacterial cultures, was used. On the basis of macrorestriction analysis after PFGE, persistence of infection was shown in all patients; two cross-transmission episodes were identified in the nosocomial as well as in the familiar environment. PCR-ribotyping proved to be useful for a DNA-based identification test, suitable for screening purposes. The rapid amplification protocol here tested is proposed to evaluate the discriminatory power of other specific target sequences in PCR-based typing assays, for epidemiologic purposes.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

Comparative analysis of amplified fragment length polymorphism and pulsed field gel electrophoresis in a hospital outbreak and subsequent endemicity of ampicillin-resistant Enterococcus faecium

Reliable molecular methods for determination of relatedness between bacterial isolates have become increasingly important to evaluate outbreaks and endemic situations with nosocomial pathogens. In the present study Simpson's index of diversity with calculated confidence intervals was used to compare amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) analysis of a hospital outbreak of ampicillin-resistant Enterococcus faecium and subsequent endemicity. The outbreak, in a Norwegian tertiary hospital, of infections caused by these enterococci started in 1995 and increased in 1996 after which the situation turned endemic. The purpose of this study was to compare the two methods in this setting and to determine the length of time during an outbreak that these methods are sufficiently valid to be of value for hospital infection control efforts. One hundred and sixty clinical isolates from urine specimens collected during the period 1995-1999 were included. The findings indicate that PFGE and AFLP are equally discriminative and could in this setting be used for typing purposes over the whole 5-year period.     

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.     

Cost-Effectiveness and Efficacy of spa,SCCmec, and PVL Genotyping of Methicillin-Resistant Staphylococcus aureus as Compared to Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA) surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab) for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa), Staphylococcal cassette chromosome (SCCmec), and Panton-Valentine leukocidin (PVL) typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type.     

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.     

Genotyping of Campylobacter coli isolated from humans and retail meats using multilocus sequence typing and pulsed-field gel electrophoresis

Aims:  To determine the antimicrobial resistant profiles and clonality of Campylobacter coli isolated from clinically ill humans and retail meats.
Methods and Results:  A total of 98 C. coli isolates (20 from humans and 78 from retail meats) were phenotypically characterized. Antimicrobial susceptibility testing was done using agar dilution method for ciprofloxacin, gentamicin, erythromycin and doxycycline. Seventy C. coli isolates including humans ( n  = 20) and retail meats ( n  = 50) were genotyped by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Resistance to ciprofloxacin was found in 29% and 15% of isolates from retail meats and humans. We observed 61 PFGE profiles using two enzymes ( Sma I, Kpn I) with an Index of discrimination of 0·99, whereas MLST generated 37 sequence types. Two clonal complexes were identified with 58 (82%) C. coli isolates clustered in the ST-828 complex.
Conclusions:  Resistance to ciprofloxacin and erythromycin was identified in C. coli obtained from retail meats and ill humans. PFGE typing of C. coli isolates was more discriminatory than MLST. Grouping of C. coli isolates (82%) by MLST in ST-828 clonal complex indicates a common ancestry.
Significance and Impact of the Study:  A high frequency of resistance found to ciprofloxacin and erythromycin is concerning from food safety perspective. PFGE using single or double restriction enzymes was found to be more discriminatory than MLST for genotyping C. coli . Overall, the C. coli populations recovered from humans and retail meats were genotypically diverse.     

Spread of a single clone Acinetobacter baumannii strain in an intensive care unit of a teaching hospital in Turkey

Acinetobacter baumannii is an important nosocomial pathogen, especially in immunocomprimised patients and those hospitalized in intensive care units. After the first isolation of A. baumannii strains from the bronchial aspirates of two patients in the intensive care unit (ICU) of our hospital as a pure culture, screening studies were performed to define possible source(s). A. baumannii strains isolated from bronchial aspirates and blood cultures of the patients in ICU were collected as a possible part of the outbreak. A total of 23 screening samples collected from equipment (7), hands (4) and gloves (2) of the staff, and from ten different body regions of the patients in the ICU were cultured. Antimicrobial susceptibility test of the isolates was performed by the standardized disk-diffusion method. All isolates were subtyped by antibiogram, arbitrarily primed polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE) typing methods. A total of 26 A. baumannii strains including eight clinical and 18 screening isolates were identified. All isolates were susceptible only to meropenem, tobramycin, and imipenem. There was at least a 96% resistance rate to the other antibiotics tested. Antibiogram typing showed that 24 of the 26 isolates were epidemiologically related, two were unique. AP-PCR yielded two types, one of which had 21 isolates, the other had five. PFGE fingerprinting revealed that all isolates were clonally related, including four closely related and 22 indistinguishable strains. Based on the results of PFGE which has been accepted as a reference method it can be concluded that A. baumannii strains isolated from our intensive care unit originated from a single type of strain.     

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).     

Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson's index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.     

Molecular fingerprinting of Campylobacter and Arcobacter isolated from chicken and water.

The potential of a fingerprinting method based on the single-enzyme amplified fragment length polymorphism (s-AFLP) technique was evaluated for its efficacy in detecting foodborne Campylobacter and Arcobacter species. Campylobacter and Arcobacter isolates from chicken and water samples were subjected to s-AFLP and pulsed-field gel electrophoresis (PFGE) profiling. Molecular typing revealed a high degree of heterogeneity. AFLP was found to be appropriate for differentiating minimal genomic variations, which makes this technique a valuable tool for the identification of isolates. PFGE was effective in showing epidemiological relationships among closely related isolates. Either technique allowed the discrimination of A. butzleri from A. cryaerophilus and A. skirrowii. When used together, s-AFLP and PFGE can be applied to determine taxonomic and epidemiological relationships among campylobacteria.     

Molecular analysis of Klebsiella pneumoniae strains isolated in pediatric wards by ribotyping, pulsed field gel electrophoresis and antimicrobial susceptibilities

The purpose of this study was to investigate the usefulness of different molecular typing techniques in the surveillance and control of the spread of extended-spectrum-beta-lactamase-(ESBL) producing Klebsiella pneumoniae in the pediatric department of the "Agostino Gemelli" hospital of the Catholic University in Rome, over a period of nine months. The strains were characterized by ribotyping using HindIII as restriction enzyme and pulsed field gel electrophoresis (PFGE) using XbaI as endonuclease. Sixty six K. pneumoniae clinical strains were isolated during this period, the first 32 were isolated in the summer of 1998. Among these first isolates, ribotyping generated 26 different patterns whereas PFGE produced 16 patterns. The remaining 34 strains were isolated during January and April 1999 and all of them were ESBL producers. Ribotyping clustered the strains into 6 patterns whereas PFGE generated only 3 patterns. PCR revealed the presence in 10 isolates of both bla(TEM) and bla(SHV) genes and 24 strains carried only the bla(SHV) gene. In our experience ribotyping revealed a higher power of differentiation with respect to PFGE and was of great help in the surveillance of the infection.