Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit.

Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in critically ill neonates and immunocompromised patients. Numerous methods have been proposed for typing. We used pulsed-field gel electrophoresis (PFGE) typing to analyze an outbreak in a neonatal intensive care unit (NICU). We included 23 patient isolates from an outbreak (March to July 1995), and 10 patient isolates from different wards during the same time period. PFGE of whole-cell DNA digested by SpeI was used as a marker of strain identity. The most common presentation of the infection was sepsis in 18 of 23 (78%) neonates. Only four different biotypes were identified; biotype A8d accounted for 84% of the strains. PFGE typing revealed two clones responsible for two different clonal strain dissemination outbreaks from March to July, with 24 patient isolates being pattern A and 4 patient isolates being pattern E. PFGE typing suggests cross transmission between patients in the NICU and other wards. The isolates from 5 other patients showed distinct PFGE patterns. Extensive investigation and cultures failed to identify any environmental or staff reservoir of S. marcescens. This is one of the first reports applying PFGE to the study of S. marcescens, and this method was a useful marker of strain identity. PFGE typing distinguished strains which appeared to be the same by biotyping.     

Use of pulsed-field gel electrophoresis to investigate a pseudo-outbreak of Bacillus cereus in a pediatric unit.

Bacillus cereus is a well-known cause of food poisoning. It also causes rare systemic infections, usually in immunocompromised patients. Dissemination of this species in hospitals had been reported. Most of these episodes were pseudo-outbreaks and were usually secondary to equipment or environmental contamination. We report here on the use of pulsed-field gel electrophoresis (PFGE) to analyze a pseudo-outbreak of B. cereus in a pediatric unit. Different restriction endonucleases had been tested, and SmaI was found to give the best result for PFGE. Among the 26 clinical isolates of B. cereus and the type strain of the species, 15 distinct PFGE patterns were distinguished. PFGE after DNA macrorestriction with SmaI could clearly differentiate between the epidemiologically related isolates and the unrelated isolates. Because the same epidemic strain of B. cereus was isolated from the settle plates which were exposed near the outlet of the ventilation system, the source of this pseudo-outbreak was suspected to be the unit's air filtration system. This is one of the first reports of the application of PFGE to the study of B. cereus, and this method is useful for epidemiological investigation.     

Use of pulsed-field gel electrophoresis for investigation of an outbreak ofClostridium difficile infection among geriatric patients

A six-month outbreak ofClostridium difficile infection among elderly residents of a middle-term-care facility was investigated. Pulsed-field gel electrophoresis was used to genotype 22 outbreak strains and 30 epidemiologically unrelated strains. A prospective case-control study was conducted to identify risk factors for epidemicClostridium difficile-associated diarrhea. All epidemiologically unrelatedClostridium difficile strains of the same serogroup could be differentiated by their DNA patterns with two restriction enzymes (Smal andKspl). Among clustered strains, two epidemic serogroups (C and K) were identified. Two different DNA patterns were identified among serogroup C strains and three among serogroup K strains. Multivariate analysis showed that the risk ofClostridium difficile infection increased with antimicrobial chemotherapy (-lactam agents and pristinamycin) and the presence of a feeding tube. This study confirms the high discriminative power of restriction fragment length polymorphism analysis by pulsed-field gel electrophoresis to describeClostridium difficile epidemiology. The typing results confirm that infection was principally exogenous in this outbreak. Furthermore, they indicate the need to improve all measures limiting transmission of infection.     

Use of pulsed-field gel electrophoresis for epidemiological study of Bordetella pertussis in a whooping cough outbreak.

We used pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with XbaI to determine the distribution of different Bordetella pertussis strains from clinical isolates obtained during a large whooping cough outbreak that occurred in Alberta, Canada, from December 1989 to May 1991. Our initial study analyzed 28 clinical isolates, 14 from the city of Edmonton and 1 from each of 14 northern Alberta towns. These clinical isolates were randomly chosen over the course of the 18-month outbreak. The DNA profiles were more heterogeneous than anticipated and caused concern that PFGE was too sensitive a technique to characterize strains. Further analysis showed that this was not the case, as clusters of similar PFGE patterns were observed in strains isolated from the same outlying town. Identical PFGE patterns were also seen in clinical strains obtained from different members of the same family. Two PFGE pattern types, a and b, predominated in the outbreak, accounting overall for 44 of 70 B. pertussis strains tested. Results from isolates from outlying towns, however, indicated involvement of local strains rather than a single, highly infectious strain in the whooping cough outbreak in Alberta.     

Nosocomial outbreak of nitrate-negative Serratia marcescens infections.

Bacteremia due to multiply-antibiotic-resistant Serratia marcescens occurred within 1 week in four patients who were in adjacent beds in an intensive care unit. The strains were serotyped as O14:H12 and were nitrate negative. This unusual biochemical marker was useful in the investigation of the outbreak.     

Epidemiological study by pulsed-field gel electrophoresis of an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a geriatric hospital.

Twelve cases of infections caused by extended-spectrum beta-lactamase (ESBla)-producing Klebsiella pneumoniae were reported between August 1991 and March 1993 in the Geriatric Department of the Nimes University Hospital, where these bacterial had not been previously isolated. Restriction profiles of total genomic DNAs cleaved by XbaI and SpeI were compared by pulsed-field gel electrophoresis. The strains that were tested included the 12 isolates from K. pneumoniae-infected patients, strains recovered from rectal swabs of asymptomatic patients in the same ward, and strains isolated in other hospitals in Nîmes at the same time. The restriction profiles of the 12 isolates and those recovered from asymptomatic patients in the same ward were very similar. Over a period of more than 1 year, extended-spectrum beta-lactamases were not detected in K. pneumoniae isolates with restriction patterns different from that of the epidemic strain. It seems, therefore, that there was no transfer of a plasmid or a gene coding for ESBla to strains of K. pneumoniae that were different from the epidemic strain. At the same time, ESBla-producing K. pneumoniae isolates exhibiting restriction endonuclease profiles very different from that of the epidemic strain were isolated from other hospitals in Nîmes. None of these strains caused an outbreak. Pulsed-field gel electrophoresis, which allows precise characterization of strains beyond the species level, is a useful tool for studying the ESBla-producing K. pneumoniae strains involved in nosocomial outbreaks.     

Epidemiological typing of methicillin-resistant Staphylococcus aureus outbreak isolates by pulsed-field gel electrophoresis and antibiogram

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens. In April 1997, there were five MRSA-infected patients among 16 patients in the Neonatal Intensive Care Unit (NICU), Seoul National University Hospital, which is a tertiary-care hospital with 1,500 beds. The infections had spread from twin patients with MRSA who had transferred from Hospital C. MRSA was isolated from the axilla of 15 (94%) of the 16 patients, including the two patients with obvious infections. Three (19%) of 16 doctors and nine (30%) of 30 nurses had MRSA colonization of the anterior nares. Six different PFGE patterns (A through F) were identified in the 53 isolates of MRSA tested. Twelve of 13 isolates from infected sites of five patients showed pattern F. Three MRSA strains obtained from hospital C showed closely or possibly related pattern F. MRSA of type F was isolated from three of 16 patients' axilla, and one of 3 doctors' and three of 30 nurses' nasal swabs. The antibiogram code for 12 of 13 MRSA isolates from five infected patients was 66,754. PFGE patterns of these isolates were either F, F1, F2 or Fa. Only one of three strains isolated from clinical specimens of patients in Hospital C showed the antibiogram code 66754, although they were all PFGE types F1 and Fa. In conclusion, the presumptive sources of the outbreak of MRSA infection in NICU were the twin patients transferred from hospital C. Antibiogram correlated reasonably well to the PFGE type. An effective notification system is needed when a MRSA-infected patient is transferred to another hospital to control the spread of the infection.     

Use of pulsed-field gel electrophoresis for investigation of hospital outbreaks of Acinetobacter baumannii.

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii.     

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.     

Use of molecular typing to study the epidemiology of Serratia marcescens.

Although Serratia marcescens is a well-known nosocomial pathogen, investigation of its hospital ecology has been limited by the lack of available typing techniques. During an investigation of the occurrence of this organism in a neonatal intensive care unit, we evaluated a number of such techniques. Using a selective medium, we conducted prospective surveillance of neonatal rectal colonization and environmental contamination with S. marcescens. In 8 months of surveillance, 5.1% (20 of 394) of the infants admitted to the unit became colonized. Most sink surfaces and drains were also culture positive. Differences between isolates could not be detected in biotypes from a commercial identification system (MicroScan) or by antibiograms, total protein fingerprints, or plasmid profiles. Serogrouping and genomic DNA restriction endonuclease analysis revealed the presence of six strains that colonized infants and a similar number of environmental strains. These two methods were concordant, with the exception that genomic DNA analysis demonstrated lack of relatedness between some strains within the same serogroup. DNA restriction endonuclease analysis was practical and reliable. The differences this method detected between environmental and neonatal strains provided strong evidence that the environment was not an important reservoir for S. marcescens in our neonatal intensive care unit.     

Epidemic outbreak of Serratia marcescens infection in a cardiac surgery unit.

Between 2 February and 16 April 1985, an outbreak of Serratia marcescens infection involving 10 male patients occurred in a cardiac surgery unit. All the patients had surgical wound infection, five also had osteomyelitis (four sternal, one costal), and another had peritonitis secondary to peritoneal dialysis. Three patients had concomitant bacteremia. All Serratia strains isolated produced a cherry-red pigment, and all had the same biochemical and antibiotic susceptibility pattern. An intensive search for the origin of the outbreak was initially unsuccessful, and it proved impossible to isolate S. marcescens from cultures of numerous samples taken from hospital personnel and from the environment. The fact that all patients were male and had been shaved for surgery by the same team of barbers led us to investigate the shaving procedures. We finally isolated a strain of pigmented S. marcescens, corresponding to that involved in the outbreak, from samples taken from the hands and equipment of the barbers. After suitable action had been taken, the epidemic terminated.     

Subspecies typing of Vibrio parahaemolyticus by pulsed-field gel electrophoresis.

Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan, and other costal regions. We report on the development of a pulsed-field gel electrophoresis (PFGE) method for the molecular typing of this pathogen. Genomic DNA was digested with SfiI, and the fragments were resolved on 1% agarose with a contour-clamped homogeneous electric field apparatus set at 190 V and a pulse time of 3 to 80 s. A total of 130 selected isolates obtained from outbreaks during 1993 and 1994 on Taiwan were also characterized by this PFGE method. These isolates were grouped into 14 PFGE types which consisted on one to six patterns, and a total of 39 patterns were identified. Most of these domestic clinical isolates could be clustered into several major types (types A, B, C, and G). These major types showed relatively low degrees of similarity to several foreign strains and other domestic but environmental strains. Strain CCRC12863, which originated from Japan, was close to the group consisting of F, G, and H PFGE types, suggesting a clonal relationship between this Japanese strain and other domestic isolates.     

Subtyping of Haemophilus influenzae strains by pulsed-field gel electrophoresis.

A total of 200 isolates of Haemophilus influenzae were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of H. influenzae demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive H. influenzae disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with H. influenzae infection revealed that the strain that caused the H. influenzae infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of H. influenzae infection, even when the invasive disease is caused by serotype b strains.     

Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis.

A disadvantage of genotyping bacterial strains by pulsed-field gel electrophoresis is that the procedure requires up to 6 days to complete. We modified a standard pulsed-field gel electrophoresis method (B.E. Murray, K.V. Singh, J.D. Health, B.R. Sharma, and G.M. Weinstock, J.Clin. Microbiol. 28:2059-2063, 1990) so that it could be completed in less than 3 days. We successfully applied this method to the analysis of a variety of gram-positive and gram-negative bacteria.     

Optimisation of Candida albicans typing by pulsed-field gel electrophoresis.

Six strains of Candida albicans were subjected to pulsed-field gel electrophoresis (PFGE) using the CHEF-DRIII system (BioRad). Hansenula mingei YB-4662-VIA and Saccharomyces cerevisiae YNN 295 (BioRad) were used as size markers (1.05-3.13 and 0.22-2.2 megabase pairs [Mbp] respectively) for comparison of DNA molecules. The DNAs were resolved by a three-block protocol with pulse times of 120 s for 24 h, 240 s for 36 h and 300 s for 17 h. The voltage was set at 4.5 V/cm for the first two blocks and 4.0 V/cm for the final block. PFGE was carried out under these conditions using different agarose concentrations, types and concentrations of buffer, temperatures, and sizes of agarose gel plug. The resolution and mobility of DNAs were affected by some of these variables. Separation of C. albicans by PFGE was optimal at 12 degrees C with 1.0 x Tris-borate-EDTA (TBE) buffer using 1.2% agarose. Resolution of banding patterns was dependent on size of DNA plug used.     

Genomic study of Rickettsia akari by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of SmaI-, EagI-, and BssHII-digested DNA was used to perform restriction fragment length polymorphism analysis of Rickettsia akari strains isolated from humans, rodents, and mites in the United States and Ukraine. Although some differences in biological and serological characteristics were present between strains, the genomic studies demonstrated a high degree of intraspecies homogeneity of R. akari isolates. Our results confirm the value of pulsed-field gel electrophoresis-restriction fragment length polymorphism analysis for the identification of species of rickettsiae.     

Serratia marcescens meningitis.

A case of Serratia marcescens meningitis in a 66-year-old man is reported. The infection occurred 4 weeks after apparently successful otic surgery, and a nidus of infection in the middle ear was established at autopsy. This is the second case of S. marcescens meningitis following ear surgery reported in the English-language literature.     

Analysis of strains of Campylobacter fetus by pulsed-field gel electrophoresis.

Campylobacter fetus chromosomal DNA from 21 strains was analyzed by pulsed-field gel electrophoresis. The fingerprint patterns generated with SmaI and SalI were distinctive. Using the profiles obtained by pulsed-field gel electrophoresis, we established the phylogenetic dendrogram of C. fetus to identify the genetic relationship of the strains.     

致病性钩端螺旋体脉冲场凝胶电泳图谱分型研究

目的 建立我国15群15型钩端螺旋体标准菌株染色体DNA脉冲场凝胶电泳图谱,进行致病性钩体的分子遗传学分类鉴定。方法 采用低熔点琼脂糖凝胶块直接纯化钩体菌完整的染色体DNA,用限制性内切酶NotI消化后,进行脉冲场凝胶电泳分离。结果 从50-1000kb范围的DNA片段得到很好的分离效果。我国常见15群15型国内标准菌株各具特异性脉冲场凝胶电泳图谱。结论 脉冲场凝胶电泳图谱可对钩体菌进行遗传学分类鉴定,这在钩体病分子流行病学研究中具有重要意义。