Pulsed-field Gel Electrophoresis

Purpose: To describe the One-Day (24-26 hour) Standardized Laboratory Protocol for Molecular Subtyping of Vibrio cholera and Vibrio parahaemolyticus by Pulsed-field Gel Electrophoresis (PFGE).Scope: To provide the PulseNet participants with the same procedure for performing PFGE of Vibrio cholerae and Vibrio parahaemolyticus, thus ensuring inter-laboratory comparability of the generated results.PNL06 Last Updated April 2013vibrio_pfge_protocol-508c.pd

Pulsed-field gel electrophoresis studies of Rhizobiaceae

The migration properties of circular, covalently closed (ccc) and linear DNAs were studied in pulsed-field gel electrophoresis as a model system for resolving large plasmids in the Rhizobiaceae. The effects of varying pulse time and agarose concentration on the electrophoretic mobility cccDNAs, ranging from 2 kilobase pairs (kb) to 16 kb, was investigated. We used both field inversion gel electrophoresis (FIGE) and electrophoresis in contour-clamped homogeneous electric fields (CHEF); both ccc and linear molecules had a minimum mobility as a function of pulse time in a CHEF apparatus. Linear and cccDNAs of the same size were affected differently by pulse time. The electrophoretic mobility of cccDNAs was pulse-time dependent in both electrophoretic systems;We used pulsed-field gel electrophoresis (PFG) and statistical analysis of Rhizobiaceae DNA sequences from GENBANK to identify rare-cutting restriction endonucleases. By using FIGE and CHEF, the genome sizes of Rhizobium meliloti and Bradyrhizobium japonicum were calculated. Comparisons were made between the genomes of free-living and bacteroid forms. The stability of genomic fingerprints produced by rare-cutting enzymes was studied on B. japonicum DNA plugs. It seems likely that large-scale DNA rearrangements do not occur during B. japonicum bacteroid development, after dedifferentiation, or by maintenance on rich media;FIGE and digestion with rare-cutting restriction enzymes were used to compare the genomes of B. japonicum field isolates from the 123 serocluster, as well as serotype strains USDA 123, USDA 127, and USDA 129. Genomic fingerprints (or field inversion gel electrophoretic types (FIGETs)), produced by four restriction enzymes, showed a high degree of diversity in the sizes of restriction fragments. Isolates could be classified according to FIGETs, and this classification was correlated with a serological classification. Hybridization with a nifHD gene probe to FIGE gels of B. japonicum DNA revealed that restriction fragment length polymorphisms (RFLPs) existed within members of serocluster 123 and provided further evidence of the relatedness between members of serogroups 123 and 127

Diversity in Acinetobacter baumannii isolates from paediatric cancer patients in Egypt

Acinetobacter baumannii is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. It is increasingly reported as a multidrug-resistant organism, which is alarming because of its capability to resist all available classes of antibiotics including carbapenems. The aim of this study was to examine the genetic and epidemiological diversity of A. baumannii isolates from paediatric cancer patients in Egypt, by sequencing the intrinsic blaOXA -51-like gene, genotyping by pulsed-field gel electrophoresis and multi-locus sequence typing in addition to identifying the carbapenem-resistance mechanism. Results showed a large diversity within the isolates, with eight different blaOXA -51-like genes, seven novel sequence types and only 28% similarity by pulsed-field gel electrophoresis. All three acquired class-D carbapenemases (OXA-23, OXA-40 and OXA-58) were also identified among these strains correlating with resistance to carbapenems. In addition, we report the first identification of ISAba2 upstream of blaOXA -51-like contributing to high-level carbapenem resistance. This indicates the presence of several clones of A. baumannii in the hospitals and illustrates the large genetic and epidemiological diversity found in Egyptian strains

Pulsed field gel electrophoresis of group A streptococci

Here we describe the protocols to perform PFGE analysis of chromosomal DNA from the bacterial species Streptococcus pyogenes (group A streptococcus, GAS) after digestion with the restriction enzyme SmaI. Large parts of the procedures are suitable for application to DNA digested with other restriction enzymes as well. We have put an effort to present extensions to solve possible limitations to the discriminatory power of the method in the specific case of S. pyogenes

Lactococcus lactis subsp. lactis infection in waterfowl: first confirmation in animals.

We report the first description, confirmed by bacteriologic and molecular (polymerase chain reaction and pulsed-field gel electrophoresis) analysis, of an infection in animals caused by Lactococcus lactis subsp. lactis, affecting waterfowl

Molecular Investigation of Tularemia Outbreaks, Spain, 1997–2008

Tularemia outbreaks occurred in northwestern Spain in 1997–1998 and 2007–2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002–00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection.project PEP 2009/1422 of the Junta de Castilla y León (Spain)

A case of bovine raw milk contamination with Listeria monocytogenes

peer-reviewedDuring routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.Teagasc Walsh Fellowship; Irish Department of Agriculture, Fisheries and Food, Food Institutional Research Measure (Irish Microbial Risk Assessment Network project); European Union (EU), 6th Framework Programme (BIOTRACER project)

Melioidosis Outbreak after Typhoon, Southern Taiwan

From July through September 2005, shortly after a typhoon, 40 cases of Burkholderia pseudomallei infection (melioidosis) were identified in southern Taiwan. Two genotypes that had been present in 2000 were identified by pulsed-field gel electrophoresis. Such a case cluster confirms that melioidosis is endemic to Taiwan

Listeria monocytogenes in Five Sardinian Swine Slaughterhouses: Prevalence, Serotype, and Genotype Characterization

In a 3-year study (2008 to 2011) to estimate the prevalence and the contamination sources of Listeria monocytogenes in pork meat in Sardinia, Italy, 211 samples were collected from five Sardinian swine slaughterhouses: 171 samples from slaughtered pigs and 40 from the slaughterhouse environment. Fifty L. monocytogenes isolates were characterized by PCR-based serotyping, presence of virulence-associated genes, and pulsed-field gel electrophoresis restriction analysis. The overall prevalence of L. monocytogenes was 33% in swine carcasses, 7% in cecal material, 23% on meat contact surfaces, and 25% on noncontact surfaces. Only two serotypes were detected: 1/2c (78%) and 1/2a (22%). In all, based on the presence of virulence-associated genes, eight pathogenic profiles were detected. Only 42% of all isolates carried the full complement of virulence-associated genes and were allotted to profile 1. Six pulsed-field gel electrophoresis profiles persisted in the slaughterhouses; restriction profiles appeared to be specific to each plant

Human-to-Dog Transmission of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) was cultured from the nose of a healthy dog whose owner was colonized with MRSA while she worked in a Dutch nursing home. Pulsed-field gel electrophoresis and typing of the staphylococcal chromosome cassette mec (SCCmec) region showed that both MRSA strains were identical