Pulsed-field Gel Electrophoresis Banding Patterns Research Articles

Bacterial dynamics during the spontaneous fermentation of cassava dough in gari production

The microbial dynamics and diversity during solid substrate fermentation of cassava, a case study of gari production in West Africa, was investigated. The 16S rDNA gene sequence analysis of the PCR-Denaturing Gradient Gel Electrophoresis (PCR–DGGE) analysis of microbial community DNA and Pulsed Field Gel Electrophoresis (PFGE) of selected isolates as well as culturing techniques using different selective media were used to monitor the bacterial dynamics during cassava fermentation. The V3 variable region of the 16S gene was analyzed and the closest relatives of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus acidophilus and Lactobacillus casei were identified by sequencing of the DGGE band amplimers. The DGGE amplimers also revealed the succession and dynamics of LAB; there was a progressive increase in their population proportional to the fermentation period. The analysis of the PFGE band patterns showed that five diverse species of LAB were involved in the fermentation. The representative isolate of each of the PFGE clusters was phenotypicaly identified as L. plantarum, L. fermentum and Leuconostoc mesenteroides by the API 50 CHL sugar fermentation profile. These combinations of parameters identified heterofermentative LAB as bacteria that initiated the fermentation, reduced the pH below four and increased the acidity of the fermentation mash. Information such as this is relevant for the development of starter cultures and predictability of the process for traditional fermented foods and to aid their intermediate and large scale production.

Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157 in Austria

Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in childhood. Shiga toxins are considered to be the most important virulence factor of EHEC strains. Non-sorbitol-fermenting EHEC O157:H7 is still the most prevalent serotype isolated worldwide; however, sorbitol-fermenting (SF) EHEC O157:H- (H- indicates nonmotility) strains are increasingly reported. Thirteen SF EHEC O157:H- strains (11 of human origin, two from animals) were detected in Austria between 2002 and 2008. Among the 11 human cases, seven suffered from HUS, two from diarrhea and the remaining two were asymptomatic. Seven of the cases were identified in patients living in or visiting (in one case) the province Salzburg, four were in patients from the province Vorarlberg. Three outbreaks with no more than three persons involved were detected, the other four cases occurred sporadically. The pulsed-field gel-electrophoresis banding patterns of the 13 SF EHEC O157:H- isolates were grouped into three distinct clusters (groups 1, 2 and 3). Strains of the three outbreaks were identical (except for one outbreak strain with one band difference) within each outbreak. In comparison, the Bavarian epidemic strain showed a pattern different from all SF O157:H- strains isolated in Austria. For effective detection of SF EHEC O157:H-, screening for Shiga toxins by ELISA and/or Shiga toxin genes by PCR is absolutely necessary; screening on the basis of phenotypic characteristics such as sorbitol-non-fermentation is not sufficient. Typing methods relying solely on investigation of O157 will detect these strains but should nevertheless also be avoided, so that the prevalent non-O157 strains causing HUS are not missed.

Epidemiological Analysis of Shiga Toxin-producing E. coli Isolated in Gwangju, Korea, by Pulse-field Gel Electrophoresis

In Gwangju, Korea, over the last 4 years, human gastrointestinal infection caused by shiga toxin-producing E. coli (STEC) increased. The aim of this study was to ascertain the genetic relatedness of STEC strains by pulsed-field gel electrophoresis (PFGE), as no data on the molecular epidemiology of STEC in Gwangju has yet been published. The PFGE banding patterns were defined for 62 of the 67 STEC strains isolated from cattle and human. There were 11 clonal types in the 11 STEC strains of cattle origin. Among the 11 STEC strains from asymptomatic person, four O91 strains were 100% similarity in band profiles. In the STEC strains isolated from diarrhea patients, same serogroups were grouped to the same cluster; O111 stains were 89.5% similarity, O157 strains 80%, O26 strains 81.5%, and O103 strains 91% similarity, respectively. In conclusion, this is the first report that a large collection of STEC strains from Korea has been analyzed, and a high degree of diversity was observed among the strains analyzed by this technique. PFGE analysis revealed that the strains isolated from human and cattle were closely related within serotypes, and it was useful for epidemiological analysis of STEC. The importance and usefulness of active laboratory surveillance of STEC such as PFGE should be recommended.

Characterization of Erysipelothrix rhusiopathiae isolates from poultry, pigs, emus, the poultry red mite and other animals

Erysipelothrix rhusiopathiae is the causative agent of erysipelas in mammals and birds, especially pigs and poultry. In order to investigate the suitability of different subtyping methods for genetic and phenotypic similarities among Swedish isolates of the organism, 45 isolates from poultry ( n = 23), pigs ( n = 17), emus ( n = 2) and the poultry red mite Dermanyssus gallinae ( n = 3) were investigated by serotyping, pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Sequence analysis of the 16S rRNA gene was performed on eleven isolates from nine animal species. The results indicated a random scattering of serotypes throughout the dendrogram based on PFGE banding patterns following SmaI digestion. In three cases, isolates with an identical PFGE pattern were of differing serotypes. No differentiation into subgroups by antimicrobial susceptibility testing by broth microdilution was possible as results were similar for all isolates. The Minimum Inhibitory Concentrations for most antimicrobials, including penicillin and oxytetracycline, were low. The 16S rRNA gene sequences (1443 nts) from eight of eleven selected isolates of Erysipelothrix spp. were identical to that of the type strain E. rhusiopathiae ATCC 19414 T. The other three isolates differed from the type strain by two or three nucleotides. While this method may be useful for identification of Erysipelothrix spp., it is unsuitable for epidemiological investigations. Similarities in PFGE banding patterns between isolates from chickens and mites supported the hypothesis that D. gallinae may act as a reservoir and vector for E. rhusiopathiae. Further PFGE studies on E. rhusiopathiae isolates are appropriate to investigate the epidemiology of poultry erysipelas.

Molecular typing of Vibrio cholerae O1 isolates from Thailand by pulsed-field gel electrophoresis.

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future.

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

BackgroundThe soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined.Methods and FindingsWe undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Twelve PFGE types and nine sequence types (STs) were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively), only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93). Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone.ConclusionsWe discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

Relationship between pathogenicity for humans and stx genotype in Shiga toxin-producing Escherichia coli serotype O157

To examine the reason why people infected with Shiga toxin (Stx) producing Escherichia coli (STEC) O157 strains develop varying clinical manifestations, 65 STEC O157 isolates originating from 64 different occurrences of infection in Miyazaki Prefecture in 2001-2003 and their 79 infected individuals were analyzed by stx genotyping, quantitative analysis of reversed passive latex agglutination (RPLA), genomic DNA analysis using pulsed-field gel electrophoresis (PFGE), and clinical manifestations. The isolates were found to carry the following stx genes: stx2vha alone (60.0%), stx1/stx2 (27.7%), stx1/stx2vha (6.1%), stx2 alone (3.1%), and stx2/stx2vha (3.1%). No strain carried the stx1 gene alone. STEC strains carrying stx2 were more frequently associated with clinical manifestations of hemolytic-uremic syndrome (HUS) or bloody diarrhea than those carrying stx2vha. Clusters of PFGE banding patterns were correlated well with the stx genotypes. We conclude that stx genotype is one of the important factors of clinical outcome of STEC O157 infection and that pathogenicity for humans was higher in the stx2 genotype strains than in the stx2vha genotype strains, as reported previously by other researchers. Further, we newly found that four clusters identified by PFGE using restriction enzyme XbaI, stx genotypes and clinical manifestations were well correlated with each other.

Genotypes, Serotypes, and Antibiotic Resistance Profiles of Salmonella Isolated from Commercial North Carolina Turkey Farms

This study was designed to determine the serotypes, genotypes, and antibiotic resistance (AbR) patterns of 42 Salmonella isolates recovered from either fecal or litter samples of 12 commercial turkey farms across two seasons (summer and winter) and two ages (3 and 19 weeks). Isolates were serotyped on the basis of the Kauffmann-White scheme. Genotyping was done by restriction digestion of cDNA (XbaI) and subsequent pulsed-field gel electrophoresis (PFGE). The AbR was determined with Sensititre susceptibility plates. Serovar Kentucky was the most prevalent serotype (26%), followed by Senftenberg (19%), Muenster (17%), Mbandaka (10%), Javiana (7%), Hadar (5%), Heidelberg (5%), 8,(20):nonmotile (5%), Agona (2%), Infantis (2%), and 4,12:r:− (2%). Serovars Kentucky, Heidelberg, Hadar, and 8,(20):nonmotile were isolated only from the 19-week-old bird samples, whereas Senftenberg and Muenster were isolated only from the young birds (3 weeks old). Isolates within any one serotype showed minor PFGE banding pattern differences, but dendogram analysis indicated that sequence variability between serotypes was more significant than within serotypes. Isolates were resistant to tetracycline (86%), sulfisoxazole (71%), streptomycin (64%), gentamicin (41%), ampicillin (36%), kanamycin (26%), sulfamethoxazole-trimethoprim (7%), nalidixic acid (5%), cefoxitin (2%), and ceftiofur (2%). One isolate (Muenster) was resistant to nine antibiotics (2%), and the others were resistant to six (7%), five (12%), four (10%), three (21%), two (24%), and one (10%) antibiotic. Only two isolates (5%) were susceptible to all antibiotics tested. The AbR patterns were affected by age; on average, strains recovered from young birds were resistant to more than four drugs compared with fewer than three in older birds (P < 0.05). This study showed that Salmonella enterica subsp. enterica serotypes, genotypes and AbR patterns were affected by bird age but not by season or farm.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from regional hospitals in Trinidad and Tobago

Methicillin-resistant Staphylococcus aureus (MRSA), first reported in a British hospital in the early 1960s, has now reached global proportions. Geographic spread of one or several MRSA clones in a city, country, and even among countries and continents has been identified by molecular techniques. We sought to determine whether clonal spread of MRSA has occurred in Trinidad and Tobago from all MRSA isolates collected between 2000 and 2001. Clinical isolates of MRSA from three major hospitals in Trinidad and Tobago were identified by standard laboratory methods and analyzed using multiplex polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE) after SmaI digestion. There was a 12.8% prevalence of MRSA in three major regional hospitals in Trinidad and Tobago. All 60 randomly selected MRSA strains from these hospitals produced similar PFGE banding patterns, suggesting a genetic relatedness among strains and that they belonged to a single clonal family. All isolates were negative for the Panton-Valentine leukocidin gene (pvl). These strains shared a PFGE banding pattern approximately (96%) the same as a Canadian strain called CMRSA-6 in the Canadian National Microbiology Laboratory database. We conclude that only one major PFGE genotype of MRSA clone is circulating among the three major regional hospitals in Trinidad and Tobago suggesting one of three possible scenarios of microevolution: (1) all were from the dissemination of a single epidemic MRSA clone prevailing in these hospitals in Trinidad and Tobago; or (2) MRSA in Trinidad and Tobago is evolving more slowly than in other countries; or (3) that if other MRSA clones have been present in Trinidad and Tobago, they have not persisted.

Genetic surveillance of endemic bovine Salmonella Infantis infection

BackgroundSalmonella serovar Infantis is endemic in Finnish food-producing animals since the 1970s. The purpose of this study was to describe the molecular epidemiology of the infection in cattle during 1985–2005, to follow the persistence of the feed-related outbreak strain from 1995 in the cattle population, and to analyse the stability of XbaI-banding patterns in individual herds during long-lasting infections.MethodsSalmonella Infantis isolates from 478 cattle herds (n = 588), covering 73% of the subclinically or clinically infected herds, were typed by pulsed-field gel electrophoresis (PFGE) using XbaI. DNA fragments larger than 125 kb were counted in PFGE types because of high plasmid background. Ribotyping and IS200-typing with BanI-digested DNA were done on 57 selected isolates.ResultsThe isolates associated with the infection consisted of 51 PFGE types with genetic similarity (F value) between 0.58 and 0.95. From 1985 to 2003, the major type appeared on 68% of the farms. The three most common types, with F values of 0.90 to 0.95, accounted for 80% of the isolates. Only 17% of the isolates had F values below 0.80, and 1% below 0.70. Ribotyping and IS200-typing classified 89% of the analysed isolates into the major ribotype and IS200 type combination, and the rest fell into closely related types. Analysis of successive isolates from 142 herds revealed changes in XbaI-banding patterns in 21% of the herds with two analysed isolates and in 38% of the herds from which three or more isolates were analysed. The feed-related S. Infantis genotype from the 1995 outbreak had disappeared by 1999, at the time when the incidence of bovine salmonella, and S. Infantis in particular, strongly decreased.ConclusionThe study showed how genetic surveillance, as part of salmonella control, provides tools to follow the persistence of particular infections, and to assess the efficacy of control measures. Testing of several isolates from a herd in outbreak investigations is advisable, because minor changes in PFGE banding patterns frequently occur during long-lasting infections.

人獣感染を分子疫学的に証明し得た&lt;I&gt;Pastenrella multocida&lt;/I&gt;肺炎の1例

We report the case of a patient with pneumonia caused by Pasteurella multocida (P. multocida). An 83-year-old woman admitted for bronchiectasis, productive cough, and a high fever was found in chest radiography on admission to have a new infiltrative shadow in the left lower lung field. Chest computed tomography indicated bilateral bronchiectasis and an infiltrative lingular shadow. P. multocida was isolated from sputum samples. Biapenem and minocycline therapy ameliorated symptoms. Pulsed-field gel electrophoresis (PFGE) showed that the clinical isolate recovered was identical to the P. multocida isolate recovered from the pharyngeal swab specimen culture obtained from the patient's dog. Several years later, P. multocida subsp. gallicida isolates were recovered from human sputum and a canine pharyngeal swab specimen. PFGE confirmed these isolates to be identical and PFGE band patterns were identical to those of previous P. multocida isolates. These findings suggest that P. multocida was transmitted from the patient's dog to the patient and underscore the need to inform susceptible hosts at risk of contracting zoonotic agents about basic hygiene rules for keeping pets. PFGE data strengthened the evidence of zoonotic transmission of P. multocida through casual contact in a patient with a respiratory infection caused by this organism. PFGE should prove useful in both epidemiological tracing and preventing pasteurellosis and clarifying its pathology. Case reports of respiratory infections including pneumonia caused by P. multocida are increasing in humans, and P. multocida respiratory infections appear to occur mostly in patients with chronic respiratory disease. A history of pet exposure, even without bites or scratches, should alert the physician to P. multocida as a potential respiratory pathogen.

Long-Term Persistence of Specific Genetic Types of Mastitis-Causing Staphylococcus aureus on Three Dairies

Pulsed-field gel electrophoresis (PFGE) after SmaI digestion was used to investigate the persistence of specific genotypes of bovine mammary gland isolates of Staphylococcus aureus on 3 dairy herds. A total of 341 isolates of Staph. aureus were available from cows in 3 herds, collected over a period of 15 yr. Pulsed-field gel electrophoresis band patterns of Staph. aureus isolates were analyzed visually and with gel analysis and comparison software. Based on this analysis, isolates were classified by PFGE type. Persistence was determined as the time period from the first to the last isolation of a particular PFGE type of Staph. aureus within a herd. Specific types of mastitis-causing Staph. aureus persisted long-term on these dairies. For example, PFGE type 3 isolates persisted on farms A, B, and C for 15, 15, and 13 yr, respectively. Type 6 was found to persist for 13 yr on farm C. Despite the application of standard mastitis control practices, mastitis-causing Staph. aureus types appeared to persist long-term, as detected by PFGE, and were isolated coincident with herd problems of increased milk somatic cell counts and decreased milk production.

Heteroresistance to Colistin in Multidrug-Resistant Acinetobacter baumannii

Multidrug-resistant Acinetobacter baumannii has emerged as a significant clinical problem worldwide and colistin is being used increasingly as "salvage" therapy. MICs of colistin against A. baumannii indicate its significant activity. However, resistance to colistin in A. baumannii has been reported recently. Clonotypes of 16 clinical A. baumannii isolates and ATCC 19606 were determined by pulsed-field gel electrophoresis (PFGE), and colistin MICs were measured. The time-kill kinetics of colistin against A. baumannii ATCC 19606 and clinical isolate 6 were investigated, and population analysis profiles (PAPs) were conducted. Resistance development was investigated by serial passaging with or without exposure to colistin. Five different PFGE banding patterns were found in the clinical isolates. MICs of colistin against all isolates were within 0.25 to 2 microg/ml. Colistin showed early concentration-dependent killing, but bacterial regrowth was observed at 24 h. PAPs revealed that heteroresistance to colistin occurred in 15 of the 16 clinical isolates. Subpopulations (<0.1% from inocula of 10(8) to 10(9) CFU/ml) of ATCC 19606, and most clinical isolates grew in the presence of colistin 3 to 10 microg/ml. Four successive passages of ATCC 19606 in broth containing colistin (up to 200 microg/ml) substantially increased the proportion of the resistant subpopulations able to grow in the presence of colistin at 10 microg/ml from 0.000023 to 100%; even after 16 passages in colistin-free broth, the proportion only decreased to 2.1%. This represents the first demonstration of heterogeneous colistin-resistant A. baumannii in "colistin-susceptible" clinical isolates. Our findings give a strong warning that colistin-resistant A. baumannii may be observed more frequently due to potential suboptimal dosage regimens recommended in the product information of some products of colistin methanesulfonate.

Ophthalmic Manifestations of Infections Caused by the USA300 Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus

To report the microbiological, clinical, and pathological characteristics of community-associated methicillin-resistant Staphylococcus aureus (CAMRSA) infections of the eye and orbit. Prospective case series. Nine patients with CAMRSA infections of the eye and orbit were identified during a 6-month period at 2 tertiary care hospitals in San Francisco. Case identification was by prospective case selection and retrospective laboratory review of 549 MRSA cultures collected in the 2 hospitals. Ophthalmic microbial isolates were analyzed by pulsed-field gel electrophoresis and compared with a control CAMRSA clone (USA300). Clinical characteristics of patients infected with CAMRSA were reviewed, and all surgical specimens underwent pathological examination. Pulsed-field gel electrophoresis banding patterns of MRSA isolates, antibiotic sensitivity profiles, patient demographics, systemic and ocular complications of infection, and posttreatment visual acuities. Nine ophthalmic isolates were CAMRSA clone USA300. The infections included orbital cellulitis, endogenous endophthalmitis, panophthalmitis, lid abscesses, and septic venous thrombosis. Patients were treated with trimethoprim-sulfamethoxazole, rifampin, clindamycin, or vancomycin based on microbial sensitivity studies and severity of infection. Eight of the 9 patients had no history of hospitalization. Seven patients required hospitalization, 3 required surgery, and an additional 4 required invasive procedures. Eight patients had good visual outcomes, but 1 deteriorated to no light perception. Pathological analyses showed extensive necrosis in eyelid and orbital specimens, and disorganized atrophy bulbi in an enucleated eye. The USA300 CAMRSA clone, which carries Panton-Valentine leukocidin genes, can cause aggressive infections of the eye and orbit in hospital-naive patients. Treatment of infections often required debridement of necrotic tissues in addition to non-beta-lactam class antibiotics. In communities where CAMRSA is prevalent, ophthalmologists should obtain microbial cultures and sensitivity studies to help guide antibiotic therapy for severe ophthalmic infections.

Molecular Characterization of Escherichia coli O157:H7 Hide Contamination Routes: Feedlot to Harvest

This study was conducted to identify the origin of Escherichia coli O157:H7 contamination on steer hides at the time of harvest. Samples were collected from the feedlot, transport trailers, and packing plant holding pens and from the colons and hides of feedlot steers. A total of 50 hide samples were positive for E. coli O157:H7 in two geographical locations: the Midwest (25 positive hides) and Southwest (25 positive hides). Hide samples were screened, and the presence of E. coli O157: H7 was confirmed. E. coli O157:H7 isolates were fingerprinted by pulsed-field gel electrophoresis and subjected to multiplex PCR procedures for amplification of E. coli O157:H7 genes stx1, stx2, eaeA, fliC, rfbEO157, and hlyA. Feedlot water trough, pen floor, feed bunk, loading chute, truck trailer side wall and floor, packing plant holding pen floor and side rail, and packing plant cattle drinking water samples were positive for E. coli O157:H7. Pulsed-field gel electrophoresis banding patterns were analyzed after classifying isolates according to the marker genes present and according to packing plant. In this study, hide samples positive for E. coli O157:H7 were traced to other E. coli O157:H7–positive hide, colon, feedlot pen floor fecal, packing plant holding pen drinking water, and transport trailer side wall samples. Links were found between packing plant side rails, feedlot loading chutes, and feedlot pens and between truck trailer, different feedlots, and colons of multiple cattle. This study is the first in which genotypic matches have been made between E. coli O157:H7 isolates obtained from transport trailer side walls and those from cattle hide samples within the packing plant.

Genes of Escherichia coli O157:H7 That Are Involved in High-Pressure Resistance

Seventeen Escherichia coli O157:H7 strains were treated with ultrahigh pressure at 500 MPa and 23 +/- 2 degrees C for 1 min. This treatment inactivated 0.6 to 3.4 log CFU/ml, depending on the strain. The diversity of these strains was confirmed by pulsed-field gel electrophoresis (PFGE) analysis, and there was no apparent association between PFGE banding patterns and pressure resistance. The pressure-resistant strain E. coli O157:H7 EC-88 (0.6-log decrease) and the pressure-sensitive strain ATCC 35150 (3.4-log decrease) were treated with a sublethal pressure (100 MPa for 15 min at 23 +/- 2 degrees C) and subjected to DNA microarray analysis using an E. coli K-12 antisense gene chip. High pressure affected the transcription of many genes involved in a variety of intracellular mechanisms of EC-88, including the stress response, the thiol-disulfide redox system, Fe-S cluster assembly, and spontaneous mutation. Twenty-four E. coli isogenic pairs with mutations in the genes regulated by the pressure treatment were treated with lethal pressures at 400 MPa and 23 +/- 2 degrees C for 5 min. The barotolerance of the mutants relative to that of the wild-type strains helped to explain the results obtained by DNA microarray analysis. This study is the first report to demonstrate that the expression of Fe-S cluster assembly proteins and the fumarate nitrate reductase regulator decreases the resistance to pressure, while sigma factor (RpoE), lipoprotein (NlpI), thioredoxin (TrxA), thioredoxin reductase (TrxB), a trehalose synthesis protein (OtsA), and a DNA-binding protein (Dps) promote barotolerance.

Epidemiological Investigation, Cleanup, and Eradication of Pullorum Disease in Adult Chickens and Ducks in Two Small-Farm Flocks

Pullorum disease causing acute septicemia and mortality in adult brown chickens was diagnosed in a small-farm chicken flock in Iowa. Also, Salmonella Pullorum was isolated from the intestine of one of four rats trapped on this index farm. Tracing movements of spent hens from the index farm resulted in identification of a second infected flock on a contact farm. Poultry on the contact farm were tested with the stained-antigen, rapid whole-blood test, and two ducks and one chicken gave positive reactions. Reactors were necropsied and cultures of appropriate tissues resulted in isolation of Salmonella Pullorum from one duck and the chicken. Pulsed-field gel electrophoresis banding patterns of Salmonella Pullorum isolates from chickens on the index and contact farms, the duck, and the rat demonstrated that all isolates were genetically very similar. Both flocks were quarantined and depopulated and a detailed flock cleanup plan was created for both farms. After extensive cleaning and disinfection procedures were completed on the index farm, environmental monitoring and bioassays of trapped mice were conducted. Negative cultures of environmental swabs and trapped mice and negative blood tests of all birds conducted 4 mo after placement of a new flock on the index farm demonstrated that cleaning and disinfection methods used in this outbreak had successfully eliminated Salmonella Pullorum organisms from this farm.

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

A modified pulsed-field gel electrophoresis (PFGE) protocol for subtyping previously non-PFGE typeable isolates of Clostridium difficile polymerase chain reaction ribotype 001

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to 50 isolates of the UK epidemic strain of Clostridium difficile, polymerase chain reaction (PCR) ribotype 001, to develop a PFGE-based subtyping scheme. This protocol overcame the inherent DNA degradation problems associated with typing this strain of C. difficile by this method, and whole genomic digestion with SmaI restriction enzyme yielded seven distinct and reproducible PFGE banding patterns. Modified PFGE is an appropriate method for subtyping C. difficile PCR ribotype 001 that could be used to improve epidemiological investigations.

Vancomycin-resistant enterococcus (VRE) carriage and infection in intensive care units.

From July, 1997, through December, 2001, patients who were admitted to intensive care units (ICUs) were enrolled in the study of vancomycin resistance enterococcus (VRE) colonization. Among 4,538 patients admitted to the ICUs, 363 (8.0%) patients were found to have positive culture of VRE at the day of admission to the ICUs and 453 (10.0%) of patients were negative to the first day of admission but became colonized with VRE during the stay in ICU. Among 816 patients, 9 (1.1%) with VRE isolated from sterile sites were selected for further analysis. Pulsed-field gel electrophoresis (PFGE) revealed a total of four PFGE banding patterns in the colonized and infected Enterococcus faecium isolates. Six of nine 9 were found to have an identical PFGE type Ia, suggesting the circulation of an endemic strain. All of these type Ia isolates also contained two potential virulence genes, the esp and hly genes and were first identified in Asia. After the further typing of 540 isolates that were randomly selected from each month, the endemic strain was not identified before the first patient was colonized and infected with this strain in November, 1998, but was isolated from other ICU patients during each month thereafter throughout the remainder of the study period. Although colonization of VRE is the first step toward infection, a low infection rate was observed, except in patients with prolonged hospitalization and severe illness. Use of the isolation room and reminders regarding hand hygiene failed to prevent the circulation of endemic strain. Thus, the SHEA guideline (Muto et al., Infect. Control Hosp. Epidemiol. 2003;24:362-386) for preventing nosocomial transmission of VRE should be enforced.