Pulsed-field Gel Electrophoresis Characterization Research Articles

PCR–Restriction Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis Characterization of Listeria monocytogenes Isolates from Ready-to-Eat Foods, the Food Processing Environment, and Clinical Samples in South Africa

Listeria monocytogenes is a ubiquitous, intracellular foodborne pathogen that is responsible for invasive listeriosis. The ability of L. monocytogenes to cause disease has some correlation with the serotypes of a specific lineage group, making the identification of lineage groups important for epidemiological analysis. The development of typing methods to link the strains of L. monocytogenes to an outbreak of listeriosis would help minimize the spread of the disease. The aim of this study was to design a PCR-restriction fragment length polymorphism (RFLP) method to differentiate between the lineage groups of L. monocytogenes. PCR-amplified fragments of the hly gene for 12 serotypes of L. monocytogenes were sequenced, aligned, and analyzed with the BioEdit program, and single nucleotide polymorphisms (SNPs) within regions of this gene were identified. Because of the difficulty in acquiring a serotype 4ab reference strain, this serotype was not included in this study. We tested the specificity and accuracy of the PCR-RFLP method on these L. monocytogenes reference strains and validated the method with 172 L. monocytogenes strains recovered from humans, food, and the food processing environment in 2000 to 2002 and 2008 to 2010 from regions within South Africa. PCR-RFLP analysis applied in this study placed L. monocytogenes serotypes into one of three lineage groups based on the sequence differences and SNPs within each lineage group. The SNPs were conserved in a region where RFLP analysis could be applied for a distinction between L. monocytogenes lineage groups.

Clinical Isolates of Acinetobacter baumannii From Tehran Hospitals: Pulsed-field Gel Electrophoresis Characterization, Clonal Lineages, Antibiotic Susceptibility, and Biofilm-forming Ability

Background: Acinetobacter baumannii is known as a potential pathogen in hospitals and is responsible for the dramatic increase in carbapenem resistance in Iran in the recent years. Objectives: The current study aimed at determining the genetic association of the isolates by the pulsed-field gel electrophoresis (PFGE) technique, identify international clones, evaluate biofilm formation ability and its relationship with antibiotic resistance. Methods: In the current study, a total of 48 A. baumannii isolates were collected from 2 hospitals in Tehran, Iran, from 2010 to 2012. Isolates were subjected to antimicrobial susceptibility testing, determination of carbapenemase encoding genes, biofilm formation, and genetic relationships analysis. Results: The obtained results demonstrated that the rate of resistance to carbapenem, meropenem, imipenem, and doripenem was 76%. The carbapenemase-encoding gene bla(OXA-23-like) was found in 32 isolates, while bla(OXA-40-like) (bla(OXA-24-like)), bla(OXA-58-like), bla(VIM-type) and bla(IMP-type) were found in 11, 1, 19. and 5 isolates, respectively. When the lineage of the isolates was evaluated by the multiplex polymerase chain reaction (PCR), it was found that 28 isolates belonged to group 1 and 8 isolates to group 2. None of the isolates belonged to group 3. Twelve isolates could not be typed by this method. The study findings interestingly demonstrated that 13 isolates showed no biofilm formation. Data of biofilm formation also demonstrated that 28, 4, and 3 remaining isolates had weak, moderate, and strong biofilm formation, respectively. The pulsed field gel electrophoresis result revealed 11 unique clones. Conclusions: International clone 1 was the most commonly identified clone in the current study. This clone was mostly associated with bla(OXA-23-like) gene; therefore, 64% of the isolates in this clone possessed bla(OXA-23-like) gene. Keywords Author Keywords:Acinetobacter baumannii; Electrophoresis; Gel; Pulsed-Field; Biofilm

Phenogenotypic Characterization of Escherichia coli O157:H7 Strains Isolated from Cattle at Slaughter.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 was isolated from 30 (4%) of 744 cattle hide swab samples collected at Estonian slaughterhouses within a 3-year monitoring program of zoonotic pathogens. The isolates were characterized by determining the presence of STEC main virulence factors, the antimicrobial resistance profiles, and the genetic relatedness by pulsed-field gel electrophoresis (PFGE). Thirteen strains carried the stx2 gene alone and 17 both the stx1 and stx2 genes. The most frequently detected stx subtype was stx2c, occurring alone (n = 12) or in combination with subtype stx1a (n = 13). All isolates harbored the intimin-coding eae gene and produced enterohemolysin. Twelve isolates (40%) showed resistance to at least one of the 14 antimicrobials and the isolates were predominantly resistant to streptomycin, sulfamethoxazole, and ampicillin. No extended-spectrum beta-lactamase-producing isolates were detected. PFGE characterization of the isolates showed an overall similarity higher than 75%, and four clusters based on 100% similarity were revealed.

The Survey of Cronobacter spp. (formerly Enterbacter sakazakii) in Infant and Follow-up Powdered Formula in China in 2012

ObjectiveTo determine Cronobacter spp. contamination in infant and follow-up powdered formula in China. MethodsAll of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI and SpeI restriction enzymes. ResultsCronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90% (10/1011) and 1.18% (15/1271), respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns. ConclusionThe current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process.

Clinical isolates of Acinetobacter baumannii from a Portuguese hospital: PFGE characterization, antibiotic susceptibility and biofilm-forming ability.

Acinetobacter baumannii is an emerging pathogen associated with nosocomial infections that in addition has shown an increasing resistance to antibiotics. In this work the genetic diversity of A. baumannii isolates from a Portuguese hospital, their antibiotic resistance profiles and ability to form biofilms was studied. Seventy-nine clinical A. baumannii isolates were characterized by pulsed-field gel electrophoresis (PFGE) with 9 different PFGE profiles being obtained. Concerning the antimicrobial susceptibility, all A. baumannii isolates were resistant to 12 of the 17 tested antibiotics and classified as multidrug-resistant (MDR). In addition, 74.7% of the isolates showed biofilm formation ability, however no statistical significance with antibiotic resistance was observed. In contrast, urine samples isolates were more likely to form biofilms than strains isolated from other sources. Our findings highlight the high number of MDR A. baumannii isolates and the importance of the formation of biofilms as a potential virulence factor.

Pulsed-Field Gel Electrophoresis characterization of Listeria monocytogenes isolates from cheese manufacturing plants in São Paulo, Brazil

This study aimed to evaluate the occurrence of Listeria monocytogenes in cheese and in the environment of three small-scale dairy plants (A, B, C) located in the Northern region state of São Paulo, Brazil, and to characterize the isolates using conventional serotyping and PFGE. A total of 393 samples were collected and analyzed from October 2008 to September 2009. From these, 136 came from dairy plant A, where only L. seeligeri was isolated. In dairy plant B, 136 samples were analyzed, and L. innocua, L. seeligeri and L. welshimeri were isolated together with L. monocytogenes. In dairy plant C, 121 samples were analyzed, and L. monocytogenes and L. innocua were isolated. Cheese from dairy plants B and C were contaminated with Listeria spp, with L. innocua being found in Minas frescal cheese from both dairy plants, and L. innocua and L. monocytogenes in Prato cheese from dairy plant C. A total of 85L. monocytogenes isolates were classified in 3 serotypes: 1/2b, 1/2c, and 4b, with predominance of serotype 4b in both dairy plants. The 85 isolates found in the dairy plants were characterized by genomic macrorestriction using ApaI and AscI with Pulsed Field Gel Electrophoresis (PFGE). Macrorestriction yielded 30 different pulsotypes. The presence of indistinguishable profiles repeatedly isolated during a 12-month period indicated the persistence of L. monocytogenes in dairy plants B and C, which were more than 100km away from each other. Brine used in dairy plant C contained more than one L. monocytogenes lineage. The routes of contamination were identified in plants B and C, and highlighted the importance of using molecular techniques and serotyping to track L. monocytogenes sources of contamination, distribution, and routes of contamination in dairy plants, and to develop improved control strategies for L. monocytogenes in dairy plants and dairy products.

Staphylococcus aureus Outbreak Investigation of an Illinois Bakery

AbstractOver 100 individuals were sickened after ingesting an assortment of desserts linked to four staphylococcal food poisoning outbreaks leading investigators to products manufactured by an Illinois bakery. The investigative team identified substantial deviations from the current Good Manufacturing Practice Regulations, 21 CFR Part 110, during multiple visits to the bakery. A total of 299 environmental swabs and 16 raw ingredients were collected for bacteriological analysis. Staphylococcus aureus isolates recovered from these samples were evaluated using either serological or polymerase chain reaction methods for the identification of staphylococcal enterotoxins SEA‐SEE, SEG‐SEI, SE‐like SElJ‐SElU or their respective genes, and for Panton–Valentine leukocidin cytotoxin. A raw ingredient and 16% of the environmental samples revealed the presence of enterotoxigenic S. aureus capable of producing diverse combinations of toxins. Antimicrobial susceptibility testing found that all S. aureus isolates were resistant to one or more agent(s). Pulsed field gel electrophoresis characterization of the isolates identified six pattern types.Practical ApplicationsThis Staphylococcus aureus outbreak investigation revealed the prevalence and diversity of antimicrobial‐resistant strains of S. aureus carrying both classical staphylococcal enterotoxins (SEs) and genes for nonclassical SE and SE‐like enterotoxins found in the environment of an Illinois bakery. In this occurrence, nonclassical SE and SE‐like enterotoxin serotypes were coupled with classical SEs, and therefore detectable by commercially available kits. This outbreak investigation demonstrated the abundance of the nonclassical SE and SE‐like enterotoxin serotypes in the environment and the anticipated need for new screening method requirements.

Pulsed-field gel electrophoresis characterization of Bordetella pertussis clinical isolates circulating in Turkey in 2001–2009

Our purpose was to evaluate the natural epidemiological history of circulating Bordetella pertussis clinical isolates in Turkey, comparing isolates by means of pulsed-field gel electrophoresis (PFGE) profiles according to years, geographic regions, vaccination status, and demographic characteristics. We analyzed genotypically a collection of 92 clinical isolates recovered during the period 2001-2009 at the National Pertussis Reference Laboratory by PFGE. A total of 61 genotypes were identified among the 92 isolates. Fifteen of 61 genotypes were a cluster including 46 isolates, and the remaining 46 genotypes were unique. The clustering rate was 50% (46/92). The size of the cluster varied from 2 to 14 clinical isolates. There was no association between clustering rates and age, gender, or quarterly season. The clustering rate was significantly higher in 2006. When the isolates were grouped according to similarity coefficient higher than 85%, 89 (96.7%) of the 92 isolates were clonally related. There was one major group including 65.2% of the isolates mainly observed. This is the first study on the molecular characterization of B. pertussis isolates in Turkey. We consider that this study lays a good foundation for further monitoring of the circulating B. pertussis clinical isolates in Turkey.

Genetic diversity of Listeria monocytogenes strains from a high-prevalence dairy farm.

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm ("farm A") revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm ("farm B"). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.

Reference system for characterization of Bordetella pertussis pulsed-field gel electrophoresis profiles.

Pulsed-field gel electrophoresis (PFGE) has been used as an epidemiological tool for surveillance studies of Bordetella pertussis since the early 1990s. To date there is no standardized procedure for comparison of results, and therefore it has been difficult to directly compare PFGE results between laboratories. We propose a profile-based reference system for PFGE characterization of B. pertussis strain variation and to establish traceability of B. pertussis PFGE results. We initially suggest 35 Swedish reference strains as reference material for PFGE traceability. This reference material is deposited at the Culture Collection of the University of Gothenburg, Gothenburg, Sweden. Altogether, 1,810 Swedish clinical isolates from between 1970 and 2003 were studied, together with the Swedish Pw vaccine strain, six reference strains, and two U.S. isolates. Our system provides evidence that profiles obtained by using only one enzyme, i.e., XbaI, give enough data to analyze the epidemiological relationship between them. Characterization with one enzyme is far less labor intensive, yielding results in half the time than when a two-enzyme procedure is used. Also, we can see that there is a correlation between PFGE profile and pertactin type. One common PFGE profile, BpSR11 (n = 455), showed 100% prn2 and 100% Fim3 when analyzed for pertactin type and serotype. On the other hand, strains with the same profile may express various serotypes when isolated over longer periods of time. Subculturing of the same isolate eight times or lyophilization caused no change in PFGE profile.

Pulsed-Field Gel Electrophoresis Characterization of Shiga Toxin–Producing Escherichia coli O157 from Hides of Cattle at Slaughter

Contamination of the brisket areas of the hides of healthy adult cattle with Shiga toxin–producing Escherichia coli O157 at slaughter in England was studied. In total, 73 cattle consignments comprising 584 animals delivered to one abattoir over 3 days during 1 week in July 2001 were studied: 26 cattle consignments arriving on Monday, 32 consignments arriving on Wednesday, and 15 consignments arriving on Friday. Consignment sizes ranged from 1 to 23 animals, with a mean consignment size of 8. The hide of the first animal to be slaughtered in each consignment was sampled by using a sponge swab moistened with 0.85% saline to rub an unmeasured brisket (ventral) area (ca. 30 by 30 cm). The process of isolating E. coli O157 from the swabs consisted of enrichment, screening with immunoprecipitation assay kits, and immunomagnetic separation. E. coli O157 was found on 24 of 73 (32.9%) cattle hides examined, and 21 of these 24 isolates produced Shiga toxins. The 24 E. coli O157 isolates produced six different pulsed-field gel electrophoresis profiles, and 18 (75%) of the isolates were of one prevalent clone. The high prevalence of one E. coli O157 clone on the hides of cattle at slaughter could be due to a high prevalence of that clone on the 18 farms involved (not investigated in the current study), in the postfarm transport or lairage environments, or both. Since the lairage environment, but not the farm of origin or the postfarm transport vehicle, was a factor common to all 18 cattle consignments, it could have played an important role in spreading the prevalent E. coli O157 clone to the cattle hides. Lairage pen floors and the stunning box floor were identified as the most probable sites along the unloading-to-slaughter route at which the brisket areas of cattle hides could become contaminated.

PCR-based DNA fingerprinting (REP-PCR, AP-PCR) and pulsed-field gel electrophoresis characterization of a nosocomial outbreak caused by imipenem- and meropenem-resistant Acinetobacter baumannii

To demonstrate the usefulness of REP-PCR and AP-PCR on molecular typing of A. baumannii isolates. From February to November 1997, 29 inpatients at Ramón y Cajal Hospital, Madrid-23 in five intensive care units (ICUs) and six at two different medical departments-were either colonized or infected with imipenem- and meropenem-resistant Acinetobacter baumannii (IMRAB) strains (MICs of 64-256 mg/L). A wide antibiotic multiresistance profile was observed with IMRAB strains, and only tobramycin, sulbactam and colistin displayed valuable activity. For typing IMRAB isolates, repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) and arbitrary primer sequence-based polymerase chain reaction (AP-PCR) methods were used and compared with pulsed-field gel electrophoresis (PFGE) as reference technique. For comparative purposes, 30 imipenem- and meropenem-susceptible A. baumannii (IMSAB) strains isolated before, during and after the outbreak were included in this study. The molecular typing results showed that the outbreak was caused by a single IMRAB strain (genotype 1). On the other hand, seven different genotypes were observed in the pre-, at- and post-outbreak strains tested by REP-PCR. Regarding AP-PCR, three of four at-outbreak IMSAB strains were indistinguishable from the IMRAB profile. Thus, with AP-PCR, only six genotypes were obtained, apart from the IMRAB genotype. Under our experimental conditions, REP-PCR had a higher discriminatory power than AP-PCR, with PFGE as reference technique. The REP-PCR technique is a useful and expeditious method for the epidemiologic characterization of A. baumannii nosocomial outbreaks, the results being comparable to those obtained with the PFGE technique.