Use Of Pulsed-field Gel Electrophoresis Research Articles

Use of Pulsed-Field Gel Electrophoresis To Monitor a Five-Strain Mixture of Listeria monocytogenes in Frankfurter Packages

In a previous study, the viability of a five-strain mixture of Listeria monocytogenes (including Scott A [serotype 4b, clinical isolate], 101M [serotype 4b, beef-pork sausage isolate], F6854 [serotype 1/2a, turkey frankfurter isolate], H7776 [serotype 4b, frankfurter isolate], and MFS-2 [serotype 1/2a, pork plant isolate]) was monitored during refrigerated storage of frankfurters prepared with and without 3.0% added potassium lactate. Throughout a 90-day period of storage at 4°C, the initial inoculum level of 20 CFU per package remained relatively constant in packages containing frankfurters prepared with potassium lactate, but pathogen counts increased to 4.6 log10 CFU in packages containing frankfurters prepared without added potassium lactate. To determine which of the five strains persisted under these conditions, randomly selected colonies obtained after 28 and 90 days of refrigerated storage of frankfurters were analyzed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI to generate distinct banding patterns for each of the five strains. Then, with the use of PFGE as a tool for identification, the percentages of the strains on days 28 and 90 of the growth study were compared. In the absence of any added potassium lactate in the product, 43% of the 58 isolates recovered on day 28 were identified as strain Scott A, 12% were identified as strain 101M, 22% were identified as strain F6854, 10% were identified as strain H7776, and 12% were identified as strain MFS-2. However, by day 90, an appreciable number (83%) of the 60 isolates analyzed were identified as strain MFS-2. In packages containing frankfurters formulated with 3.0% potassium lactate, all five strains were present at frequencies of 5 to 36% among the 19 isolates tested on day 28; however, by day 90, strain MFS-2 made up the statistical majority (63%) of the 27 isolates tested. The results of this study indicate that strain MFS-2, a serotype 1/2a isolate recovered from a pork processing plant, was more persistent than strains Scott A, 101M, F6854, or H7776 during the extended refrigerated storage of frankfurters.

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of salmonella isolates obtained from the carcasses and feces of swine at slaughter.

Salmonella enterica isolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmed Salmonella isolates from the 84 Salmonella-positive samples obtained from the previous study. A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups. The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively. In addition, multiple isolates were obtained from 67 of the 84 Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples. Both carcass and fecal isolates of Salmonella were recovered from 13 swine, resulting in "matched" samples. Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated with Salmonella pulsotypes that were not isolated from the feces of the same animal. Conversely, from 6 of the 13 (46%) matched animals, Salmonella clonal types were isolated from the feces that were not isolated from the carcass of the same animal. These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses. In addition, these results indicate that the examination of multiple Salmonella isolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing.

Epidemiologic study of results of pulsed-field gel electrophoresis of isolates of Rhodococcus equi obtained from horses and horse farms.

To compare isolates of Rhodococcus equi on the basis of geographic source and virulence status by use of pulsed-field gel electrophoresis (PFGE). 290 isolates of R equi (218 virulent isolates from foals and 72 avirulent isolates from feces, soil, and respiratory tract samples) obtained between 1985 and 2000 from horses and horse farms from 4 countries. DNA from isolates was digested with the restriction enzyme Asel and tested by use of PFGE. Products were analyzed for similarities in banding patterns by use of dendrograms. A similarity matrix was constructed for isolates, and the matrix was tested for nonrandom distributions of similarity values with respect to groupings of interest. There was little grouping of isolates on the basis of country, virulence status, or region within Texas. Isolates of R equi were generally < 80% similar, as determined by use of PFGE. Isolates from the same farm generally were rarely of the same strain. Considerable chromosomal variability exists among isolates of R equiobtained from the same farm, sites withinTexas, or among countries from various continents. Only rarely will it be possible to link infections to a given site or region on the basis of analysis of isolates by use of PFGE of chromosomal DNA.

Tandem amplification of a 28-kilobase region from the Yersinia enterocolitica chromosome containing the blaA gene.

Most Yersinia enterocolitica strains are resistant to beta-lactam antibiotics due to the production of one or two chromosomally encoded beta-lactamases. Strain Y56 is a Y. enterocolitica O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A beta-lactamase. To select mutants with increased levels of resistance to beta-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 micro g of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a blaA-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the blaA gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the blaA locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event.

Epidemiology of vancomycin-resistant Enterococcus faecium under a selective isolation policy at an urban county hospital

Background: We report our experience in a county hospital with the use of selective contact isolation for patients with vancomycin-resistant Enterococcus faecium (VREF). About 12% of patients with VREF are isolated for reasons such as draining wounds and uncontrolled diarrhea. Methods: Passive surveillance identified all inpatients (181) from 1995 to 1999 with cultures positive for VREF. Data were collected via electronic databases and from prospectively maintained infection control records. Isolates were typed with use of pulsed-field gel electrophoresis. Results: Nearly all patients (175/181) with VREF had been admitted at least 48 hours or had a history of previous hospitalization. Most patients (69%) had urine cultures positive for VREF without blood cultures positive for the organism. Only 12 of 127 (9.%) patients with complete data had VREF infection on the basis of receiving treatment and/or having more than 1 blood culture positive for VREF. After VREF became endemic, statistically significant increased prevalence was not detected via surveillance of clinical cultures nor sequential point-prevalence studies. Two major genotypes carrying vanB resistance genes were identified and persisted throughout the period studied. VREF persisted in individual patients up to 46 months. Conclusions: The number of VREF infections in this facility has been low, despite appreciable colonization, for an extended period during which selective isolation was used. (Am J Infect Control 2002;30:400-6.)

Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices.

The hypothesis tested was that dairy herds which import cattle for replacement or expansion have a higher prevalence of Staphylococcus aureus mastitis and a greater number of new Staphylococcus aureus strains enter their herds than closed herds. Fifteen commercial dairy herds were divided into four groups based on cattle importation practices. Composite foremilk samples were collected at 4-monthly intervals for 1 year from all lactating cattle. Additionally, foremilk samples were collected from cattle at parturition and skin swabs were taken from the udder of primiparous heifers. All samples were cultured for Staphylococcus aureus and isolates were strain-typed using pulsed-field gel electrophoresis. Herds that purchased replacement heifers had a higher prevalence of Staphylococcus aureus mastitis than herds that purchased lactating cattle for expansion (P = 0.02). Herds that purchased replacement heifers had more total strains of Staphylococcus aureus (P = 0.01) and more new strains (P = 0.04) enter the herd than closed herds.

A multistate outbreak of Shiga toxin-producing Escherichia coli O26:H11 infections in Germany, detected by molecular subtyping surveillance.

In the spring of 2000, a cluster of indistinguishable Shiga toxin-producing Escherichia coli (STEC) O26:H11 was identified in Germany by molecular subtyping surveillance. An investigation was prompted to identify a common source of exposure. A case subject was defined as a person having a polymerase chain reaction-confirmed STEC O26 infection between March and April 2000, irrespective of clinical signs, and whose isolate was indistinguishable from the index strain by use of pulsed-field gel electrophoresis. Eleven case subjects were found in 5 institutions that were supplied by 4 kitchens located in 3 states. The median age was 2 years (range, 2-31 years). No bloody diarrhea was reported, and 5 persons remained asymptomatic. Comparison of invoices revealed a certain type of beef ("Seemerrolle") as possible source of infection. This is, to our knowledge, the first multistate outbreak associated with a non-O157 STEC detected by laboratory-based surveillance. Molecular subtyping was pivotal, as disease occurrence was sporadic or family-related.

The molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in the United States, 1994-2000.

The genetic relatedness of 672 penicillin-resistant isolates of Streptococcus pneumoniae (PRSP) recovered during national surveillance studies conducted in the United States during the periods of 1994-1995, 1997-1998, and 1999-2000 was determined by use of pulsed-field gel electrophoresis (PFGE). Overall, 104 different PFGE types were elucidated. For all study periods combined, the 12 most prevalent PFGE types included >75% of all isolates, and 5 types were closely related to widespread clones (Spain(23F)-1, France(9V)-3, Spain(6B)-2, Tennessee(23F)-4, and Taiwan(19F)-14). From 1994-1995 to 1999-2000, 3 major PFGE types (not closely related to 16 recognized clones) increased in prevalence. Multidrug resistance was identified among 96%-100% of the isolates in 9 of 12 predominant PFGE types. The prevalence of erythromycin resistance increased within 4 major PFGE types. These observations support the hypothesis that the dominant factor in the emergence of PRSP in the United States during the 1990s has been human-to-human spread of relatively few clonal groups that harbor resistance determinants to multiple classes of antibiotics.

Routes and sources of Staphylococcus aureus transmitted to the surgical wound during cardiothoracic surgery: possibility of preventing wound contamination by use of special scrub suits.

To trace the routes of transmission and sources of Staphylococcus aureus found in the surgical wound during cardiothoracic surgery and to investigate the possibility of reducing wound contamination, with regard to total counts of bacteria and S. aureus, by wearing special scrub suits. A total of 65 elective operations for coronary artery bypass graft with or without concomitant valve replacement were investigated. All staff present in the operating room wore conventional scrub suits during 33 operations and special scrub suits during 32 operations. Bacteriological samples were taken from the hands of the scrubbed team after surgical scrub but before putting on sterile gowns and gloves and from the patients' skin (incisional area of sternum and vein harvesting area of legs) after preoperative skin preparation with chlorhexidine gluconate. Air samples were taken during operations. Bacteriological samples also were taken from the subcutaneous walls of the surgical wound just before closing the wound. Total counts of bacteria on sternal skin and wound walls (colony-forming units [CFUs]/cm2) were calculated, as well as total counts of bacteria in the air (CFUs/m3). Strains of S. aureus recovered from the different sampling sites were compared by pulsed-field gel electrophoresis (PFGE). Special scrub suits significantly reduced total counts of bacteria in air compared to conventional scrub suits (P=.002). The number of air samples in which S. aureus was found was significantly reduced by special scrub suits compared with conventional scrub suits (P=.016; relative risk, 4.4; 95% confidence interval [CI95], 1.3-14.91). By use of PFGE, it was possible to identify two cases of possible airborne transmission of S. aureus when wearing conventional scrub suits, whereas no case was found when wearing special scrub suits. When exposed to airborne S. aureus, the concomitant sternal carriage of S. aureus was a risk factor for having S. aureus in the wound. Use of tightly woven special scrub suits reduces the dispersal of total counts of bacteria and of S. aureus from staff in the operating room, thus possibly reducing the risk of airborne contamination of surgical wounds. The importance of careful preoperative disinfection of the patient's skin should be stressed.

Source and route of methicillin-resistant Staphylococcus epidermidis transmitted to the surgical wound during cardio-thoracic surgery. Possibility of preventing wound contamination by use of special scrub suits

The objective of this study was to trace the source and route of transmission of methicillin-resistantStaphylococcus epidermidis (MRSE) in the surgical wound during cardio-thoracic surgery, and to investigate the possibility of reducing wound contamination by wearing special scrub suits. In total 65 elective operations for coronary artery bypass grafting (CABG) with or without concomitant valve replacement were investigated. All staff present in the operating room wore conventional scrub suits during 33 operations and special scrub suits during 32 operations. Samples were taken from the hands of the scrubbed team after surgical scrub but before putting on sterile gowns and gloves, and from patients' skin (incisional area of sternum and vein harvesting area of legs) after preoperative skin preparation with chlorhexidine gluconate. Air samples were taken during operations. Samples were also taken from the wound just before closure. Total counts of bacteria on sternal skin and from the wound (cfu/cm2) were calculated as well as total counts of bacteria in the air (cfu/m3). Strains of MRSE recovered from the different sampling sites were compared by pulsed field gel electrophoresis (PFGE). It was found that wearing special scrub suits did not reduce the number of air-samples where MRSE was found compared with conventiona<$>l scrub suits. The risk factor most strongly associated with MRSE in the wound at the end of the operation was preoperative carriage of MRSE on sternal skin; RR 2.42 [95% CI 1.43–4.10], P= 0.021. By use of PFGE, it was possible to identify the probable source for four MRSE isolates recovered from the wound. In three cases the source was the patients own skin. Finding MRSE in air-samples, or on the hands of the scrubbed team, were not risk factors for the recovery of MRSE in the wound at the end of operation. In conclusion, with a total bacterial air count around 20cfu/m3and a low proportion of MRSE, the reduction of total air counts by use of tightly woven special scrub suits did not reduce air counts of MRSE or wound contamination with MRSE. The patients' sternal skin was the main source for wound contamination with MRSE

Use of pulsed-field gel electrophoresis and flagellin gene typing in identifying clonal groups of Campylobacter jejuni and Campylobacter coli in farm and clinical environments.

Although campylobacters have been isolated from a wide range of animal hosts, the association between campylobacters isolated from humans and animals in the farm environment is unclear. We used flagellin gene typing and pulsed-field gel electrophoresis (PFGE) to investigate the genetic diversity among isolates from animals (cattle, sheep, and turkey) in farm environments and sporadic cases of campylobacteriosis in the same geographical area. Forty-eight combined fla types were seen among the 315 Campylobacter isolates studied. Six were found in isolates from all four hosts and represented 50% of the total number of isolates. Seventy-one different SmaI PFGE macrorestriction profiles (mrps) were observed, with 86% of isolates assigned to one of 29 different mrps. Fifty-seven isolates from diverse hosts, times, and sources had an identical SmaI mrp and combined fla type. Conversely, a number of genotypes were unique to a particular host. We provide molecular evidence which suggests a link between campylobacters in the farm environment with those causing disease in the community.

Emergence of a high-level cefotaxime-resistant Streptococcus pneumoniae strain in New Zealand.

An increase in the number of cefotaxime-resistant pneumococci referred for surveillance to a central laboratory in New Zealand occurred in 1997-1998. The MIC of cefotaxime for 113 of 216 cefotaxime-resistant isolates of Streptococcus pneumoniae referred was > or = 4 mg/L. Most of the 113 isolates exhibited the same antibiotic resistance pattern and belonged to serotype 19F. To investigate the genetic relatedness of the isolates, 48 serotype 19F pneumococci with varying susceptibility to cefotaxime were further typed by macro-restriction analysis by use of pulsed-field gel electrophoresis. These results suggested that a multiresistant 19F strain of S. pneumoniae with high-level cefotaxime resistance had emerged from a pre-existing serotype 19F strain.

Use of Pulsed-Field Gel Electrophoresis in the Analysis of Recurrent Staphylococcus aureus Infections in Patients on Continuous Ambulatory Peritoneal Dialysis

Background/Aim: The purpose of this study was to evaluate pusled-field gel electrophoresis (PFGE) for distinguishing between relapse and reinfection of Staphylococcus aureus infections in patients on continuous ambulatory peritoneal dialysis (CAPD). Methods: Between July 1993 and May 1997, 4 patients with recurrent CAPD-associated infections caused by S. aureus we enrolled in this study. There were nine episodes of peritonitis, one episode of temporary double lumen catheter infection, and one episode of Hickman catheter infection. A total of eleven S. aureus isolates were collected from peritoneal fluid (n = 9) and blood (n = 2). PFGE typing was applied. Results: In our study, from PFGE typing, the 11 S. aureus isolates were classified into seven patterns. Antibiogram profiling classified only four patterns. Patient A had a reinfection by another strain of S. aureus, and patient B had three episodes of peritonitis caused by the same strain of S. aureus due to exit site infections. Patient C had two episodes of CAPD peritonitis caused by two different strains, respectively. Patient D had four episodes of S. aureus infection (three CAPD peritonitis and one bacteremia); the first two episodes of peritonitis were caused by an identical strain of S. aureus, whereas the subsequent two infections were caused by other organisms. Conclusion: PFGE has a high discriminatory power and can be an assistant method to antibiogram profiling for distinguishing relapse from reinfection in CAPD-associated peritonitis.

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis.

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

An outbreak of Campylobacter jejuni infections associated with food handler contamination: the use of pulsed-field gel electrophoresis.

In 1998, an outbreak of Campylobacter jejuni infections occurred in Kansas among persons attending a school luncheon; community cases were also reported. In a cohort study of luncheon attendees, 27 (17%) of 161 persons reported illness. Consuming gravy (relative risk [RR], 4.2; 95% confidence interval [CI], 1.5-11.7) or pineapple (RR, 2.4; 95% CI, 1.0-5.7) was associated with illness. Both foods were prepared in a kitchen that served 6 other schools where no illness was reported. A cafeteria worker at the luncheon had a diarrheal illness and was the likely source of the outbreak. The pulsed-field gel electrophoresis (PFGE) patterns of the isolates from the food handler and those of 8 lunch attendees were indistinguishable. Isolates from 4 community patients differed. This was the first use of PFGE in a Campylobacter outbreak in the United States; its use was critical in determining that community cases were not linked.

Is a second urine specimen necessary for the diagnosis of asymptomatic bacteriuria?

By use of pulse-field gel electrophoresis, we evaluated the molecular identity of 32 Escherichia coli isolates obtained in 2 consecutive urine cultures from 16 patients as part of a large study of asymptomatic bacteriuria in diabetic women and found different E. coli isolates in 7 of 16 patients, meaning that nearly half (44%) of the patients who had been previously classified as having asymptomatic bacteriuria were reinfected with a different strain.

Identification of Listeria monocytogenes contamination sources in two fresh sauce production plants by pulsed-field gel electrophoresis

Twenty-three Listeria monocytogenes strains isolated from two food-processing plants, which produce fresh sauces, were serologically characterized and tested by the mouse biological assay and molecular typing by pulsed-field gel electrophoresis (PFGE). The use of PFGE for the characterization of these L. monocytogenes strains provided, in plant A, valuable information about potential sites of cross-contamination and in plant B, a valuable insight into the presence of endemic strains. The use of highly discriminating typing techniques such as PFGE and the thorough observance of GMPs and the HACCP system can reduce the incidence of L. monocytogenes in food-processing plants.

Endemic carbapenem-resistant Acinetobacter species in Brooklyn, New York: citywide prevalence, interinstitutional spread, and relation to antibiotic usage.

Acinetobacter species are problematic nosocomial pathogens. In November 1997, pathogens isolated by microbiology laboratories were collected from 15 hospitals in Brooklyn, New York. Acinetobacter species accounted for 10% of gram-negative isolates. Only half of Acinetobacter species were susceptible to carbapenems; 11 hospitals had at least 1 isolate resistant to carbapenems. Other Acinetobacter susceptibility rates were as follows: polymyxin, 99%; amikacin, 87%; ampicillin/sulbactam, 47%; ceftazidime, 25%; and ciprofloxacin 23%. Overall, 10% were resistant to all commonly used antibiotics. Genetic analysis by use of pulsed-field gel electrophoresis of 12 carbapenem-resistant isolates revealed 4 strains that were recovered from >1 hospital, which suggests interinstitutional spread. Antibiotic usage data from 11 hospitals revealed that the use of third-generation cephalosporins was associated significantly with the percentage of carbapenem-resistant strains (P=.03). Resistant Acinetobacter species have become endemic in Brooklyn, New York. Citywide strategies that involve surveillance, infection-control practices, and the reduction of antibiotic usage may be necessary to control the spread of these pathogens.

Use of pulsed-field gel electrophoresis for investigation of a cluster of invasive group a streptococcal illness—Spokane, Washington, 1999

[Centers for Disease Control and Prevention. Use of pulsed-field gel electrophoresis for investigation of a cluster of invasive group A streptococcal illness—Spokane, Washington, 1999. MMWR Morb Mortal Wkly Rep. 1999;48:681-683.]

Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel "comet assay".

Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.