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Context.—: Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population., Objective.—: To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups., Design.—: Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content., Results.—: The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in a 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics., Conclusions.—: The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results., Competing Interests: Hall, Milligan, Kelnar, Hallmark, Ashton, Parker, Filipovic-Sadic, and Latham are employees of Asuragen and report owning company stock/options. The other authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Background Protein concentration of monoclonal immunoglobulin in plasma-cell myeloma/multiple myeloma provides an estimate of the tumor mass and allows for monitoring of the response to treatment. Accurate and reproducible estimates of the monoclonal immunoglobulin concentration are important for patient care. Objective To address the optimum method for estimation of the concentration of monoclonal immunoglobulins. Methods Serum protein electrophoresis and immunofixation electrophoresis were conducted by using the Helena SPIFE Touch instrument. Estimation of the protein concentration of monoclonal immunoglobulin in the gamma region by computer-assisted reading was compared with the reading by technologists and pathology residents, in 300 gels. The data were compared using t -testing and analysis of variance. Results Computer-generated readings had a consistent positive bias. The correlation coefficient of the average reading by technologists and residents with the computer generated value was 0.997. The average positive bias by the computer reading was 0.29 g per dL. The intercept on the regression analysis was 0.22 g per dL. The reading by the computer was significantly higher than each of the human-interpreted readings. The readings by the 3 human groups were not significantly different amongst them. The main reason for the higher reading by the computer was inclusion of a greater area on the anodal size of the peak on the densitometric scan. Conclusions Human- and computer-interpreted readings of the protein concentration of monoclonal immunoglobulin have a high degree of correlation. The consistent positive bias by the computer reading occurred due to inclusion of a greater area of the densitometric scan on the anodal side of the peak. We suggest that vendors should adjust such computer programs to provide readings comparable to those generated by expert humans. We recommend manual delineation of the monoclonal peaks for measuring the concentration of monoclonal immunoglobulins. [ABSTRACT FROM AUTHOR]

To clarify the characteristics of circulating Bordetella pertussis isolates, we used pulsed-field gel electrophoresis (PFGE) to analyze 5,262 isolates collected in the United States during 2000-2012. We found 199 PFGE profiles; 5 profiles accounted for 72% of isolates. The most common profile, CDC013, accounted for 35%-46% of isolates tested from 2000-2009; however, the proportion of isolates of this profile rapidly decreased in 2010. Profile CDC237, first seen in 2009, increased rapidly and accounted for 29% of 2012 isolates. No location bias was observed among profiles during 2000-2010, but differences were observed among isolates from different states during 2012. Predominant profiles match those observed in recent European PFGE studies. PFGE profile changes are concurrent with other recent molecular changes in B. pertussis and may be contributing to the reemergence of pertussis in the United States. Continued PFGE monitoring is critical for understanding the changing epidemiology of pertussis.

Taylorella equigenitalis is the etiologic agent of contagious equine metritis (CEM), a venereal disease of horses. A total of 82 strains of T. equigenitalis isolated in the United States were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of genomic DNA with restriction enzyme ApaI. Twenty-eight of those strains isolated from horses in the 2009 U.S. outbreak (CEM09) were further analyzed with NotI and NaeI enzymes. When ApaI alone was used for analysis, the 82 isolates clustered into 15 different genotypes that clearly defined groups of horses with known epidemiological connections. The PFGE profiles of the CEM09 isolates were indistinguishable after digestion with ApaI, NotI, and NaeI and did not match those of isolates from previous U.S. outbreaks in 1978 and 2006 or of any other isolate from the National Veterinary Services Laboratories (NVSL) culture library. Coupled with the fact that the CEM09 isolates are epidemiologically related, these results suggest a common source for the outbreak not linked to previous occurrences of CEM in the United States.

Salmonella enterica serovar Typhimurium circulating in food animal populations and carrying resistance to antimicrobial agents represents a human health risk. Recently, a new clade of S. Typhimurium, WA-TYP035/187, was reported in cattle and humans in the Pacific Northwest, United States of America. The objective of this study was to describe a possible mechanism of acquisition of expanded-spectrum cephalosporin resistance in this clade. Ceftazidime resistance increased steadily among WA-TYP035/187 isolates, from 0% (0/2) in 1999 to 77.8% (28/36) in 2006 (chi2 for linear trend, P value of <0.001). Among 112 bovine-source and 18 human-source isolates, 49 (43.8%) and 12 (66.7%) were resistant to ceftazidime, respectively. Multiple-locus variable-number tandem-repeat analysis (MLVA) and plasmid profiling suggested that resistance was acquired by multiple independent genetic events within the WA-TYP035/187 clade. Given the lack of an obvious reservoir in species other than cattle and a parallel rise in ceftiofur resistance in the bovine-specific serovar Salmonella enterica serovar Dublin in the same time frame and region, selection pressure due to the use of the expanded-spectrum cephalosporin drug ceftiofur in cattle is a likely factor driving the increasing cephalosporin resistance of WA-TYP035/187.

Salmonella enteritidis (SE) is an important cause of egg-associated outbreaks in both Europe and the United States. Phage typing has become an important epidemiologic tool in identifying the source of outbreaks. Limitations of phage typing have become apparent with wholesale egg distributors that have multiple suppliers in an area where a particular phage type is endemic. Several different molecular typing methods were evaluated for their discriminatory power to identify genetic differences among different SE phage types isolated in Europe and the United States. Pulsed-field gel electrophoresis (PFGE) identified a single DNA pattern among the different SE phage types. Comparison of the nucleotide sequence for several Salmonella virulence genes failed to identify a single nucleotide change in the gene sequences from most SE isolates, regardless of phage type. On the basis of these results, the different SE phage types appear to be genetically related or clonal. However, with primers 1283 and Opa4, it was possible to differentiate not only SE isolates from different geographic locations but those within a specific geographic locale as well by random amplified polymorphic DNA polymerase chain reaction. Any chance for discerning genetic differences among isolates will need to rely on molecular techniques other than PFGE.

Until recently, group B streptococcus, serotype V (GBS-V), was an infrequent cause of disease. It is now recognized as a significant cause of infections in both children and adults. To determine if this increase was due to the recent introduction and spread of a single clone of GBS-V, we analyzed, by pulsed-field gel electrophoresis (PFGE), the SmaI chromosomal DNA digests of 45 bacteria: 41 isolated from human infections between 1986 and 1996 in the United States, 2 from human infections in Argentina, and 2 from naturally infected mice. Seventeen patterns were found and arbitrarily designated patterns A to Q. Pattern N constituted 24 (53%) of the isolates and was found in all of the years tested and from all surveillance areas, as well as in both isolates from Argentina, and was very similar to the GBS-V isolated from a mouse. Pattern P was found in three isolates, pattern F was found in two, and the remaining patterns were found in one isolate each. We concluded that the majority of isolates of GBS-V are of one PFGE subtype and that this subtype was predominate before the increase in disease caused by GBS-V and that GBS-V disease is caused by several different subtypes.

Salmonella enterica subspecies enterica serovar Heidelberg (Salmonella Heidelberg) has caused several multistate foodborne outbreaks in the United States, largely associated with the consumption of poultry. However, a 2015-2017 multidrug-resistant (MDR) Salmonella Heidelberg outbreak was linked to contact with dairy beef calves. Traceback investigations revealed calves infected with outbreak strains of Salmonella Heidelberg exhibited symptoms of disease frequently followed by death from septicemia. To investigate virulence characteristics of Salmonella Heidelberg as a pathogen in bovine, two variants with distinct pulse-field gel electrophoresis (PFGE) patterns that differed in morbidity and mortality during the multistate outbreak were genotypically and phenotypically characterized and compared. Strain SX 245 with PFGE pattern JF6X01.0523 was identified as a dominant and highly pathogenic variant causing high morbidity and mortality in affected calves, whereas strain SX 244 with PFGE pattern JF6X01.0590 was classified as a low pathogenic variant causing less morbidity and mortality. Comparison of whole-genome sequences determined that SX 245 lacked ~200 genes present in SX 244, including genes associated with the IncI1 plasmid and phages; SX 244 lacked eight genes present in SX 245 including a second YdiV Anti-FlhC(2)FlhD(4) factor, a lysin motif domain containing protein, and a pentapeptide repeat protein. RNA-sequencing revealed fimbriaerelated, flagella-related, and chemotaxis genes had increased expression in SX 245 compared to SX 244. Furthermore, SX 245 displayed higher invasion of human and bovine epithelial cells than SX 244. These data suggest that the presence and up-regulation of genes involved in type 1 fimbriae production, flagellar regulation and biogenesis, and chemotaxis may play a role in the increased pathogenicity and host range expansion of the Salmonella Heidelberg isolates involved in the bovine-related outbreak. [ABSTRACT FROM AUTHOR]

Pulsed-field gel electrophoresis (PFGE) analysis of restriction pattern polymorphism was applied to type Clostridium difficile isolated from neonates hospitalized in a neonatal intensive care unit, and the results were compared with those of immunoblot analysis. C. difficile was isolated from fecal specimens of 41 (61%) of 67 neonates during a 5-month investigation. All of these neonates were asymptomatic. Fifty-five C. difficile isolates from 32 patients were analyzed by PFGE after digestion with SmaI and SacII endonucleases and by immunoblotting with 10 different antisera. Fifty-three of 55 isolates from 30 patients were identical by PFGE analysis after SmaI and SacII digestion and immunoblot analysis. Two isolates were different from each other and from the epidemic group by both PFGE and immunoblot analysis. All 53 epidemic isolates were nontoxigenic, while the two remaining isolates were toxigenic. These results suggest that nosocomial spread of nontoxigenic C. difficile infection in the neonatal intensive care unit and suggest that both PFGE and immunoblot are powerful typing tools for the epidemiological study of C. difficile.

Staphylococcus aureus is one of the most important pathogens causing contagious mastitis in dairy cattle worldwide. The objectives of this study were to determine if recently described S. aureus genotype B was present among previously characterized isolates from cases of bovine intramammary infection in the United States and to compare pulsed-field gel electrophoresis (PFGE) to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for typing of S. aureus strains. The hypothesis was that isolates that were previously characterized as contagious would be identified as genotype B and that the results of the two strain-typing methods would be comparable. Isolates were selected from a collection of S. aureus isolates from eight dairy farms. Mammary quarter milk somatic cell count (SCC) and N-acetyl-β-d-gluconaminidase (NAGase) activity data were known and used to evaluate strain pathogenicity. RS-PCR was performed with conventional gel electrophoresis, and PCR was used for toxin gene identification. RS-PCR patterns were associated with a specific virulence gene pattern, as previously reported. Five RS-PCR banding patterns were identified. None of the isolates were characterized as genotype B. No association between RS-PCR types and milk SCC was found; however, NAGase activity was significantly higher in milk from mammary glands infected with RS-PCR banding type 1 (RSP type 1) than in milk from those infected with RSP type 2. The discriminatory power values were 1.0 and 0.46 for PFGE and RS-PCR, respectively. These data suggest that genotype B may have a limited geographic distribution and that PFGE is more discriminatory than RS-PCR performed with conventional gel electrophoresis for typing of S. aureus isolates of bovine origin., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell's Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneupolyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible through capillary electrophoresis (CE) using fluorescence-labeled SSR markers. Twenty-four sugarcane cultivars, 12 each from India and the USA, were genetically assessed using 21 fluorescence-labeled polymorphic SSR markers. These markers primed the amplification of 213 alleles. Of these alleles, 161 were common to both Indian and US cultivars, 25 were specific to the Indian cultivars, and 27 were observed only in the US cultivars. Only 10 alleles were monomorphic. A high level of heterozygosity was observed in both Indian (82.4%) and US (91.1%) cultivars resulting in average polymorphism information content (PIC) values of 0.66 and 0.77 and marker index (MI) values of 5.07 and 5.58, respectively. Pearson correlation between PIC and MI was significant in both sets of cultivars (r = 0.58 and 0.69). UPGMA clustering separated cultivars into three distinct clusters at 59% homology level. These results propose the potential utility of six Indian cultivar-specific SSR alleles (mSSCIR3&#95;182, SMC486CG&#95;229, SMC36BUQ&#95;125, mSSCIR74&#95;216, SMC334BS&#95;154, and mSSCIR43&#95;238) in sugarcane breeding, vis a vis transporting CE-based evaluation in clone or variety identity testing, cross fidelity assessments, and genetic relatedness among species of the genus Saccharum and related genera.

Objective--To evaluate the usefulness of an antibody detection ELISA and protein electrophoresis (PE) for diagnosing Encephalitozoon cuniculi (ECUN) infection in pet rabbits. Animals--203 pet rabbits. Procedures--Serum and plasma samples from pet rabbits were submitted from veterinary clinics within the United States. Participating veterinarians completed a questionnaire that was used to classify rabbits as clinically normal (n=33), suspected of having an ECUN infection (103), or clinically abnormal but not suspected of having an ECUN infection (67). An ELISA for detection of serum or plasma IgG against ECUN was developed by use of commercially available reagents. Results of the ELISA and PE were used to detect ECUN infection. Results--A high seroprevalence of antibody against ECUN was detected in all 3 groups of rabbits. In rabbits suspected of having an ECUN infection, the mean IgG titer was 1.7 times as high as the values in the other rabbit groups. Rabbits suspected of having an ECUN infection and those that were simply clinically abnormal had a higher concentration of γ-globulins than clinically normal rabbits. This increase in globulins concentration was accompanied by a decrease in the albumin-to-globulin ratio. Results of the ELISA and PE were significantly different between clinically normal rabbits and those suspected of having an ECUN infection. Conclusions and Clinical Relevance--The combination of an ELISA and PE may aid in the diagnosis of ECUN infection in pet rabbits. Impact for Human Medicine--Because ECUN is a potential zoonotic agent, diagnostic methods for pet rabbits need to be improved to protect human health. [ABSTRACT FROM AUTHOR]

Conventional pulsed-field gel electrophoresis (PFGE) protocols, used extensively as a successful approach for subtyping many salmonellae, may be inadequate for discriminating strains sharing levels of homogeneity within the same serotype. Four additional restriction enzymes (SpeI, PacI, SfiI, and NotI), in addition to XbaI and BlnI, were used in PFGE typing of 33 Salmonella Heidelberg, 27 Salmonella Kentucky, 27 Salmonella SaintPaul, and 27 Salmonella Hadar isolates that were recovered from poultry and porcine retail meats from different states of the United States. A dendrogram derived from the combined analysis of six enzymes was highly discriminatory with a Simpson index of diversity value of over 0.950. The ratio of nodes to isolates was more than 0.75 with an average of fewer than three isolates in each polytomy for all four serotypes. Two three-enzyme combinations, SpeI/NotI/SfiI for Salmonella Heidelberg and Salmonella Hadar, and SpeI/BlnI/Sfil for Salmonella Kentucky and Salmonella SaintPaul, were found to have comparable discriminatory abilities of differentiating isolates of these Salmonella serotypes with the six-enzyme combination. The enhanced discriminatory PFGE-based subtyping scheme can be used effectively for the differentiation of strains of the four Salmonella serotypes. The findings also highlight PFGE analysis as a continued essential and informative subtyping method for source tracking and outbreak investigations of these and other Salmonella pathogens. [ABSTRACT FROM AUTHOR]

Reports on the 2003 International Symposium on Capillary Chromatography and Electrophoresis held in Las Vegas, Nevada. Highlights of the symposium; Presentation of the Marcel Golay Award in Capillary Chromatography and other awards; Conference sessions; Gas-phase separations; Liquid-phase separations.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90 degrees C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.

Molecular epidemiology analyses of the 36 clinical isolates of Pasteurella multocida from various avian hosts in Japan between 1976 to 2007 including 5 reference strains from the U.S.A., Taiwan and Indonesia were performed by employing the single-enzyme amplified fragment length polymorphism (SE-AFLP) comparison with the classical ApaI-based pulsed-field gel electrophoresis (PFGE). As the results, SE-AFLP gave 21 profiles while PFGE gave 20 profiles. The Simpson's index of diversity analysis indicated that SE-AFLP gave a high discrimination power than PFGE. This concluded that SE-AFLP is a higher discrimination power than PFGE to differentiate avian P. multocida isolates in Japan. In addition, the genetical profiles suggested that there is the evolution of somatic serotype 3 strain in the indigenous host of Japan.

The Food and Drug Administration (FDA) is announcing the availability of a guidance entitled "Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions; Annex 10: Polyacrylamide Gel Electrophoresis General Chapter. The guidance was prepared under the auspices of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The guidance provides the results of the ICH Q4B evaluation of the Polyacrylamide Gel Electrophoresis General Chapter harmonized text from each of the three pharmacopoeias (United States, European, and Japanese) represented by the Pharmacopoeial Discussion Group (PDG). The guidance conveys recognition of the three pharmacopoeial methods by the three ICH regulatory regions and provides specific information regarding the recognition. The guidance is intended to recognize the interchangeability between the local regional pharmacopoeias, thus avoiding redundant testing in favor of a common testing strategy in each regulatory region. In the Federal Register of February 21, 2008 (73 FR 9575), FDA made available a guidance on the Q4B process entitled "Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions."

We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same "unrelated" organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.

Salmonella enterica is a leading cause of food-borne illness worldwide and is also a major cause of morbidity and mortality in domestic and wild animals. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and nonhuman serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR while incorporating a 6-carboxyfluorescein-labeled universal primer to fluorescently label all amplicons. The fluorescently labeled PCR products were separated using capillary electrophoresis, and a Salmonella multiplex assay for rapid typing (SMART) code was generated for each isolate, based upon the presence or absence of PCR products generated from each target gene. Seven hundred fifty-one blind clinical isolates of Salmonella from Washington State, collected in 2007 and previously serotyped via antisera, were screened with the assay. A total of 89.6% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously determined serotype, while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to that of conventional serotyping, but at a fraction of both the cost and time required per test.

Background:Meticillin-resistant Staphylococcus pseudintermedius (MRSP) infections in companion animals are increasing and are difficult to treat. Environmental contamination with MRSP in small animal primary care hospitals may pose an exposure risk to animal patients. Methods: This longitudinal study assessed the genotypic relationships of MRSP isolated from 39 environmental samples collected from six private small animal primary care hospitals, in the north-eastern United States, between August 2018 and April 2019. Results: Of the 39 bacterial isolates, 18 unique pulsotypes were identified based on pulsed-field gel electrophoresis, including six clusters of two or more indistinguishable isolates. Single pulsotypeswere frequently detected frommultiple hand-contact and animal-contact surfaces within a hospital during a single sampling event, but detection of a single pulsotype within the same hospital on subsequent visits was infrequent. However, one pulsotype was recovered from three separate hospitals, which suggests that either MRSP transmission between hospitals may have occurred via people, animals, or fomites or that there was a dominant community strain. Conclusions: Single strains of MRSP were isolated from various hand-contact and animal-contact surfaces within hospitals, indicating the important role of humans, animals and the environment inMRSP transmission. Additionally, the detection of a single strain between hospitals and over time suggests that eitherMRSP transmission between hospitalsmay have occurred via people, animals or fomites or that there was a dominant community strain. [ABSTRACT FROM AUTHOR]

Conventional pulsed-field gel electrophoresis (PFGE) protocols, used extensively as a successful approach for subtyping many salmonellae, may be inadequate for discriminating strains sharing levels of homogeneity within the same serotype. Four additional restriction enzymes (SpeI, PacI, SfiI, and NotI), in addition to XbaI and BlnI, were used in PFGE typing of 33 Salmonella Heidelberg, 27 Salmonella Kentucky, 27 Salmonella SaintPaul, and 27 Salmonella Hadar isolates that were recovered from poultry and porcine retail meats from different states of the United States. A dendrogram derived from the combined analysis of six enzymes was highly discriminatory with a Simpson index of diversity value of over 0.950. The ratio of nodes to isolates was more than 0.75 with an average of fewer than three isolates in each polytomy for all four serotypes. Two three-enzyme combinations, SpeI/NotI/SfiI for Salmonella Heidelberg and Salmonella Hadar, and SpeI/BlnI/SfiI for Salmonella Kentucky and Salmonella SaintPaul, were found to have comparable discriminatory abilities of differentiating isolates of these Salmonella serotypes with the six-enzyme combination. The enhanced discriminatory PFGE-based subtyping scheme can be used effectively for the differentiation of strains of the four Salmonella serotypes. The findings also highlight PFGE analysis as a continued essential and informative subtyping method for source tracking and outbreak investigations of these and other Salmonella pathogens.

The genus Leptospira is composed of spirochetes that are morphologically indistinguishable and cannot be differentiated by phenotypic methods. Determining the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Pulsed-field gel electrophoresis (PFGE) of Leptospira has had limited use in few laboratories. In this study, we modified the existing PFGE protocol to reduce time to completion and developed a PFGE database using the restriction endonuclease Not I to generate PFGE profiles for the identification and evaluation of Leptospira. Reference strains (n = 206) from a collection maintained by the Centers for Disease Control and Prevention were studied. Eighty-nine percent of the serovars produced distinct patterns, and different strains of the same serovar showed profiles that were highly similar. Pulsed-field gel electrophoresis of Leptospira is a useful tool for molecular characterization of reference serovars, the investigation of potentially new species or serovars, and ultimately for the routine identification of clinical isolates.

Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

Five different serotypes of Salmonella enterica were implicated in a large outbreak linked to fresh Roma tomatoes served at gas station deli counters in Pennsylvania and nearby states during July 2004: S. enterica serotypes Javiana, Anatum, Thompson, Typhimurium, and Muenchen. One of these serotypes, Anatum, was isolated from both tomatoes and patients. Pulsed-field gel electrophoresis (PFGE) played a key role in identifying the outbreak-associated isolates and distinguishing them from unrelated sporadic isolates. It also demonstrated that the genetic fingerprints of serotype Anatum isolates derived from patients were indistinguishable from those derived from tomatoes. Rapid communication of PFGE fingerprints with other public health laboratories through the Centers for Disease Control and Prevention's PulseNet USA national molecular surveillance network for bacterial food-borne pathogens facilitated the tracking of this outbreak in other states. The work described in this report emphasizes the laboratory's role in core public health functions and services, thereby providing a highly visible example of public health in action.

Rapid and sensitive methods for accurate strain delineation are essential for monitoring and preventing transmission of methicillin-resistant Staphylococcus aureus (MRSA). Pulsed-field gel electrophoresis (PFGE) has been the standard technique for strain typing most bacterial species including MRSA. The goal of this study was to compare the performance of the DiversiLab microbial typing system (Bacterial BarCodes, Inc., Houston, TX) (rep-PCR) to that of PFGE for typing MRSA isolates from five well-defined outbreaks. The DiversiLab rep-PCR assay is a rapid, semiautomated method based on PCR amplification of specific regions between noncoding repetitive sequences in the bacterial genome. rep-PCR was performed according to the manufacturer's recommendations, and the results were analyzed and dendrograms were generated using the DiversiLab analysis software (version 2.1.66 a). PFGE was performed and interpreted according to published procedures. rep-PCR results using similarity indices (SI) of 80%, 85%, and 90% were compared to PFGE analysis. In addition, intra- and interrun reproducibility was determined for rep-PCR. Overall, correct assignment to outbreak versus nonoutbreak clusters occurred for 91 of 109 isolates (85% agreement) when using a SI of 85%. For each specific outbreak, concordance between rep-PCR and PFGE ranged from 73% to 100%. There were 18 discrepant results (17%). Fourteen isolates were unique by PFGE, but they were placed in clusters by rep-PCR; the other 4 were placed in clusters different from those assigned by PFGE. Intra- and interrun reproducibility was excellent. Times to results were 12 to 24 h for rep-PCR compared to 2 to 4 days for PFGE. Rapid, standardized results and excellent reproducibility make rep-PCR a valuable tool for use in MRSA investigations. However, since rep-PCR was less discriminatory than PFGE, we recommend that it be used to screen isolates, followed by testing isolates which share the same rep-PCR pattern with a more sensitive method, such as PFGE or multilocus sequence typing.

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.

Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to beta-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.

Bartonella henselae causes severe disease in immunocompromised individuals. B. henselae was isolated from 12 human immunodeficiency virus (HIV)-infected individuals with bacillary angiomatosis and/or peliosis hepatis and from their 15 cat contacts. Specific associations between the 2 B. henselae genotypes, individual pulsed-field gel electrophoresis (PFGE) patterns, and different clinical syndromes and pathogenicity were investigated. The role of cat contacts as the source of human infection was also examined. Three of the 4 patients with B. henselae genotype I infection, but none of the 8 patients with genotype II infection, had hepatosplenic vascular proliferative lesions (P=.018). Four of 5 human-cat pairs had closely-related PFGE patterns and concordant results by 16S rDNA typing, which strongly suggests that human infection was caused by the cat contact. These results corroborate the major role of cats in the transmission of B. henselae to humans and suggest that B. henselae genotypes may induce different pathological features in HIV-infected patients.

Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus.

To assess the genetic diversity of pneumococci causing serious disease within the United States, restriction profiles of 3 penicillin-binding protein (PBP)-gene amplicons and the dhf amplicon were examined in 241 recent sterile-site isolates from 7 population centers. This analysis provided markers useful for epidemiologic studies and was generally predictive of resistances to beta-lactam antibiotics and trimethoprim-sulfamethoxazole. Eight pulsed-field gel electrophoresis (PFGE) types, each representing 3-40 isolates, accounted for 134 of the 144 beta-lactam-resistant pneumococci (MICs >/=1 microgram/mL for penicillin, cefotaxime, or both). Five of these PFGE types contained subtypes highly related to subtypes of previously characterized pneumococcal clones. Within 4 of these PFGE types, the major composite PBP gene-dhf profile was highly related to the composite profile from the previously characterized related clone. Eight capsular serotypes were found among the 144 beta-lactam-resistant pneumococci. Divergent capsular types among isolates with identical PBP gene-dhf profiles and related PFGE types indicated several instances of capsular serotype switching.

Clostridium perfringens is a common cause of food-borne illness. The illness is characterized by profuse diarrhea and acute abdominal pain. Since the illness is usually self-limiting, many cases are undiagnosed and/or not reported. Investigations are often pursued after an outbreak involving large numbers of people in institutions, at restaurants, or at catered meals. Serotyping has been used in the past to assist epidemiologic investigations of C. perfringens outbreaks. However, serotyping reagents are not widely available, and many isolates are often untypeable with existing reagents. We developed a pulsed-field gel electrophoresis (PFGE) method for molecular subtyping of C. perfringens isolates to aid in epidemiologic investigations of food-borne outbreaks. Six restriction endonucleases (SmaI, ApaI, FspI, MluI, KspI, and XbaI) were evaluated with a select panel of C. perfringens strains. SmaI was chosen for further studies because it produced 11 to 13 well-distributed bands of 40 to approximately 1,100 kb which provided good discrimination between isolates. Seventeen distinct patterns were obtained with 62 isolates from seven outbreak investigations or control strains. In general, multiple isolates from a single individual had indistinguishable PFGE patterns. Epidemiologically unrelated isolates (outbreak or control strains) had unique patterns; isolates from different individuals within an outbreak had similar, if not identical, patterns. PFGE identifies clonal relationships of isolates which will assist epidemiologic investigations of food-borne-disease outbreaks caused by C. perfringens.

Arbitrarily primed PCR (AP-PCR) and pulsed-field gel electrophoresis (PFGE) subtyping were applied to clinical and environmental isolates from seven unrelated outbreaks of Legionnaires' disease. The patterns observed with each method matched patient isolates and the epidemiologically linked source of disease for each of the seven outbreaks. PFGE allowed more discrimination among various isolates, although AP-PCR usually gave comparable results. With both methods, certain patterns appeared to predominate in the comparison of the seven outbreaks. Of five clinical isolates not associated with the outbreaks, three gave profiles distinct from those observed in the outbreaks by both methods. This suggests that there are at least two predominant subtypes of Legionella pneumophila serogroup 1 associated with outbreaks. Investigations of outbreaks of legionellosis should employ either PFGE or AP-PCR in addition to monoclonal antibody analysis.

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (+/-1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.

In 1992 a helical microorganism associated with chronic active hepatitis and a high incidence of hepatocellular tumors was identified in the hepatic parenchyma of A/JCr mice. By using biochemical tests, phenotypic characterization, and 16S rRNA gene sequence analysis, the organism was classified as a novel Helicobacter species and named Helicobacter hepaticus. Recent surveys completed in our laboratory indicate that H. hepaticus is widespread in academic and commercial mouse colonies. The aim of this study was to examine the H. hepaticus genome by pulsed-field gel electrophoresis (PFGE) to determine the degree of genomic variation and genomic size. This technique has been used to identify significant genomic diversity among strains of Helicobacter pylori and to demonstrate only slight genomic diversity among strains of Helicobacter mustelae. Genomic DNAs from 11 isolates of H. hepaticus from the United States, Germany, France, and The Netherlands were subjected to PFGE after digestion with SmaI. Isolates from three independent sources within the United States had very similar PFGE patterns, suggesting that the genomic DNAs of these isolates are conserved. Genomic DNA isolated from a fourth source within the United States had a PFGE pattern different from those of the other U.S. isolates. Isolates obtained from Germany, France, and The Netherlands had PFGE patterns that differed markedly from those of the U.S. isolates and from one another. The use of DNA fingerprinting may be useful in subsequent epidemiological studies of H. hepaticus when the source and method of spread of this murine pathogen need to be ascertained. By PFGE, the genomic size of H. hepaticus is estimated to be roughly 1.3 Mb, which compares to 1.67 Mb for H. pylori and 1.7 Mb for H. mustelae.

Borrelia burgdorferi sensu lato is present all over the world. However, most species belonging to this complex have a limited distribution; the only exception is B. burgdorferi sensu stricto, which is encountered both in Europe and in the United States. The aim of this study was to clarify the way that B. burgdorferi sensu stricto migrated. To do this, we compared the genetic polymorphism of 33 B. burgdorferi sensu stricto strains isolated in Europe and 28 strains isolated in the United States by using both pulsed-field gel electrophoresis and arbitrarily primed PCR. Phylogenetic analysis of each of the two sets of data was performed by a genetic distance method and a parsimony method. The results were consistent and revealed that the American strains were more heterogeneous than the European strains. The hypothesis that B. burgdorferi sensu stricto was introduced to Europe from America rather than vice versa has to be considered.

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

Foods of bovine origin have been identified as sources of Escherichia coli O157:H7. Genomic DNA of E. coli O157:H7 isolates from patients (six isolates), food samples (18 isolates from ground beef and six isolates from raw milk) and calf faecal samples (31 isolates) were characterised by pulsed-field gel electrophoresis (PFGE) and plasmid DNA analysis. These isolates originated from different locations in the USA during 1992 and 1993. Twenty-one distinct genomic profiles were generated from the 61 isolates by PFGE after digestion with the endonuclease XbaI. Four genomic profiles were identified among five patient isolates and the remaining patient isolate was not typable. Five different profiles were detected amongst the isolates from ground beef, one of which was associated with 13 ground beef isolates from an outbreak in the Pacific Northwest of the USA in 1993. The PFGE profile of five calf isolates from Washington and Wisconsin was identical to the profile of the ground beef isolates from the outbreak, suggesting that these isolates were related. Similarly, one PFGE profile accounted for three isolates from calf faeces and one from ground beef. Six raw milk isolates from Georgia were indistinguishable both from each other and from one isolate from calf faeces. Fourteen genomic profiles were identified among 31 calf faecal isolates from 18 different herds in 11 states. Only five plasmid profiles were identified among the 61 isolates. PFGE was shown to be a useful typing technique for E. coli O157:H7.

Nineteen isolates of Mycobacterium haemophilum were analyzed by pulsed-field gel electrophoresis of large restriction fragments generated by digestion of chromosomal DNA with XbaI. Six patterns were observed. Twelve of 16 M. haemophilum isolates (75%) collected in the New York Metropolitan Area from 1990 to 1991 shared the same pattern, including all six isolates submitted from one hospital. Two different patterns were seen among the other four isolates. Individual isolates from Albany, N.Y., Florida, and Texas had unique patterns. Pulsed-field gel electrophoresis is the first method reported with the capability to type strains of M. haemophilum and will hopefully provide insight into the source and transmission of this emerging pathogen.

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.

The genomes of 62 North American and European Borrelia burgdorferi isolates were examined by restriction endonuclease analysis (REA), gene probe restriction fragment length polymorphism, and pulsed-field gel electrophoresis (PFGE). Hybridization of restriction fragments with the immunologically relevant 83-kDa antigen gene revealed polymorphisms and divided the isolates into three major groups. Group I included all but two of the American isolates and some of the European isolates. One of two Californian isolates (DN 127) and one Ixodes dammini isolate from New York (strain 25015), previously described as atypical, were distinct from the isolates in the three groups. Plasmid profile analysis and REA, the method with the highest level of discrimination, revealed extensive heterogeneity among isolates of the same major group. Our study demonstrates the usefulness of the 83-kDa antigen gene probe for dividing the isolates into major genogroups, whereas REA and plasmid profile analysis allow for a distinction of individual strains within these groups.

To investigate the microbiology and epidemiology of the 1,700 sporadic cases of listeriosis that occur annually in the United States, we developed a multilocus enzyme electrophoresis (MEE) typing system for Listeria monocytogenes. We studied 390 isolates by MEE. Eighty-two electrophoretic types (ETs) were defined. Two distinct clusters of ETs, ET group A (ETGA) and ET group B (ETGB), separated at a genetic distance of 0.440, were identified. Strains of ETGB were associated with perinatal listeriosis (P = 0.03). All strains of H antigen type a were in ETGA, while all strains of H antigen type b were in ETGB. Among 328 clinical isolates from cases of literiosis, 55 ETs of L. monocytogenes were defined. Thirty-four ETs were identified among 62 isolates from food products. The mean number of strains per ET (5.2) was significantly higher among clinical isolates than among food-borne isolates. Examination of isolates from outbreaks further documented the link between cases and contaminated food products. In one investigation, we found 11 different ETs, ruling out a single common source as a cause of that outbreak. By examining a large number of isolates collected over a specified time in diverse geographic locations in the United States, we have begun to establish a baseline for the study of the epidemiology of listeriosis by MEE.

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion, V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme. [ABSTRACT FROM AUTHOR]

Recently, multi-drug-resistant (MDR) Salmonella enterica subspecies enterica serovar Newport reemerged as a public and animal health problem. The antibiotic resistance of 198 isolates and the pulsed-field gel electrophoresis patterns (PFGE) of 139 isolates were determined. Serovar Newport isolates collected between 1988 and 2001 were included in the study. One hundred seventy-eight isolates were collected from the San Joaquin valley in California and came from dairy cattle clinical samples, human clinical samples, bulk tank milk samples, fecal samples from preweaned calves, and waterways. Twenty clinical isolates from humans from various regions of the United States were also included in the study. Resistance to 18 antibiotics was determined using a disk diffusion assay. PFGE patterns were determined using a single enzyme (XbaI). The PFGE and antibiogram patterns were described using cluster analysis. Although the antibiotic resistance patterns of historic (1988 to 1995) and contemporary (1999 to 2001) isolates were similar, the contemporary isolates differed from the historic isolates by being resistant to cephalosporins and florfenicol and in their general sensitivity to kanamycin and neomycin. With few exceptions, the contemporary isolates clustered together and were clearly separated from the historic isolates. One PFGE-antibiogram cluster combination was predominant for the recent isolates, which were taken from human samples from all parts of the United States, as well as in the isolates from California, indicating a rapid dissemination of this phenotypic strain. The data are consistent with the hypothesis that the reemergence of MDR serovar Newport is not simply an acquisition of further antibiotic resistance genes by the historic isolates but reflects a different genetic lineage. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus and V. parahaemolyticus , found naturally in marine and estuarine environments, are the leading cause of seafood associated gastrointestinal illness and death. Consumption of improperly cooked crabs and handling of live crabs are potential routes of exposure to pathogenic bacteria such as V. vulnificus and V. parahaemolyticus. Little information is available on serotype genetic and antimicrobial profiles of V. vulnificus and V. parahaemolyticus recovered from Maryland estuaries. The aim of the present study was to determine the serotype of V. parahaemolyticus , evaluate antimicrobial susceptibility and genetic profiles of V. vulnificus and V. parahaemolyticus isolated from water and blue crab (Callinectes sapidus) samples collected from the Maryland Coastal Bays. One hundred and fifty (150) PCR confirmed V. parahaemolyticus including 52 tdh + (pathogenic) and 129 V. vulnificus strains were tested for susceptibility to twenty (20) different antibiotics chosen by clinical usage for Vibrio species. The O serogroups were determined using an agglutination test with V. parahaemolyticus antisera. Pulsed-field gel electrophoresis (PFGE) was used for molecular subtyping to investigate the genetic diversity among tested strains. The most prevalent serotypes were O5 (33.3%), O3 (18.7%) and O1 (14.7%). More than 41% of all tested Vibrio isolates were resistant to three or more antibiotics. Cephalothin showed the highest resistance (42% and 61%), followed by cefoxitin (42% and 31%) and ceftazidime (36% and 29%) for V. vulnificus and V. parahaemolyticus , respectively. Most strains (99–100%) were susceptible to ampicillin/sulbactam, levofloxacin, piperacillin, piperacillin/tazobactam, and tetracycline. Fifty percent (50%) of the cephalothin resistant strains were crab isolates. Vibrio vulnificus and V. parahaemolyticus isolates demonstrated a high genetic diversity and 31% of V. vulnificus and 16% of V. parahaemolyticus strains were PFGE untypeable. No correlations were found between the V. parahaemolyticus serotype, pathogenicity, genetic and antimicrobial resistance profiles of both species of Vibrio. The observed high multiple drug resistance of V. vulnificus and V. parahaemolyticus from blue crab and its environment is of public health concern. Therefore, there is a need for frequent antibiotic sensitivity surveillance for Vibrio spp. [ABSTRACT FROM AUTHOR]

Presents a study which used pulsed-field gel eletrophoresis to evaluate the trends in the incidence of Bordetella pertussis in Cincinnati, Ohio, from 1989 to 1996. Methodology; Results; Discussion.

Background Mycobacterium abscessus , an emerging pathogen in healthcare settings, has rarely been associated with community outbreaks. During February–May 2013, Idaho public health officials and pediatric infectious disease physicians investigated an outbreak of M abscessus skin infections in children whose only common exposure was an indoor wading pool. Methods Healthcare providers and parents reported possible M abscessus cases. We used a standardized questionnaire to interview parents of affected children. Clinical specimens were submitted for mycobacterial examination. We conducted an environmental investigation of the pool. Microbial isolates from clinical and environmental samples were identified by sequencing polymerase chain reaction amplicons and underwent pulsed-field gel electrophoresis. Results Twelve cases were identified. Specimens from 4 of 7 children grew M abscessus or Mycobacterium abscessus/Mycobacterium chelonae. Ten (83%) of 12 children were female; median age was 3 years (range, 2 to 6 years); and all were immunocompetent. Pool maintenance did not fully comply with Idaho state rules governing pool operation. Mycobacterium abscessus / chelonae was isolated from pool equipment. Pulsed-field gel electrophoresis composite patterns were 87% similar between isolates from the pool ladder and 1 patient, and they were 90% similar between isolates from 2 patients. Environmental remediation included hyperchlorination, scrubbing and disinfection of pool surfaces, draining the pool, and replacement of worn pool materials. Conclusions Immunocompetent children acquired M abscessus cutaneous infection involving hands and feet after exposure to a wading pool. Environmental remediation and proper pool maintenance likely halted transmission. Medical and public health professionals' collaboration effectively detected and controlled an outbreak caused by an emerging recreational waterborne pathogen. [ABSTRACT FROM AUTHOR]