Search

High-resolution and efficient typing for Laribacter hongkongensis (L. hongkongensis) is essential for epidemiological investigation of such emerging foodborne pathogens. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is an innovative molecular method that shows great promise for L. hongkongensis typing. Here, we explored the CRISPR typing method by combining CRISPR1 and CRISPR2 loci to characterize a collection of 109 L. hongkongensis isolates from humans and animals and compared it to current molecular methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The results showed that all three methods have high discriminatory power (diversity index was 0.9902 for PFGE, 0.9663 for CRISPR and 0.9562 for MLST); strong congruence was observed between them (Rand index was 0.969 between CRISPR and PFGE, 0.953 between CRISPR and MLST, 0.958 between PFGE and MLST). CRISPR typing could well distinguish the isolates in the same STs or PFGE profiles, and the genetic information contained by the CRISPR array is useful for deep phylogenetic typing. We demonstrate that rapid CRISPR typing is a practical genetic fingerprinting tool with high resolution, comparable ease of use and lower cost, ability to track the source of various groups of L. hongkongensis strains and indication of genetic characteristics., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

DNA markers are used as a size reference and sample loading control during gel electrophoresis. Most markers are designed for conventional gel electrophoresis to separate DNA smaller than 20 kb. For larger molecules, pulsed-field gel electrophoresis (PFGE) marker is required. Limited PFGE markers are available because large DNA are prone to nicking and degradation, causing smeary bands. Here, we developed a robust marker based on bacterial artificial chromosomes (BACs) with bands up to 184 kb. This marker could consistently confer intense and distinct bands for accurate gel analysis in molecular biology studies, laboratory validations or clinical diagnosis., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Listeria monocytogenes is a serious human pathogen and an enduring challenge to control for the ready-to-eat food processing industry. Cost-effective tools that can be deployed by commercial or in-house laboratories to rapidly investigate and resolve contamination events in the built food processing environment are of value to the food industry. Multilocus variable number tandem-repeat analysis (MLVA) is a molecular subtyping method, which along with other same-generation methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) is being superseded in disease tracking and outbreak investigations by whole-genome sequencing (WGS). In this paper, it is demonstrated that MLVA can continue to play a valuable role as a valid, fast, simple, and cost-effective method to identify and track Listeria monocytogenes subtypes in factory environments, with the method being highly congruent with MLST. Although MLVA does not have the discriminatory power of WGS to identify truly persistent clones, with careful interpretation of results alongside isolate metadata, it remains a powerful tool in situations and locations where WGS may not be readily available to food business operators., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In order to provide more phylogenetic information of Campylobacter coli in large-scale epidemiological investigation, this work was undertaken to develop a novel genotyping method based on amplified intergenic locus polymorphism (AILP), by using pulsed-field gel electrophoresis (PFGE; using SmaI enzymes) as control. Eleven pairs of primers were selected to type C. coli strains for this purpose. A total of 68 C. coli isolates recovered from 51 retail raw chicken and 37 retail raw duck were subtyped. The Simpson's index of diversity (SID) of AILP and PFGE, as well as the adjusted Rand index (AR) and the adjusted Wallace coefficient (AW) between AILP and PFGE, were calculated. The new AILP method differentiated 68 C. coli isolates into 55 different subtypes (SID = 0.993), compared with 46 different profiles obtained from PFGE (SID = 0.980). The SID value of the AILP method was improved with the increasing number of primers, and a combination of 7 loci was selected as the optimal combination. The congruent analysis of the AILP method and PFGE showed moderate congruence between the two methods (AR = 0.462). The AW indicated that if AILP data is the available one can confidently predict the PFGE cluster. The results of this study showed that the AILP method had higher discrimination than PFGE and also allowed for significant reduction in time and cost., Competing Interests: Declaration of Competing Interest No potential conflict of interest associated with this manuscript was declared by the all authors., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We subtyped 32 Salmonella enterica strains isolated from carcasses (n=10), the environment (n=14), head meat (n=1) and viscera washing and chilling water (n=7) in provincial abattoirs with no Hazard Analysis Critical Control Point (HACCP) system from Buenos Aires, Argentina, before and after implementing improvement actions. Pulsed-field gel electrophoresis (PFGE) was carried out using the XbaI restriction enzyme. Strains belonged to six serovars, from which 10 restriction patterns were obtained (five unique patterns and five clusters). We found different clones of S. enterica serovars in the same abattoir by XbaI-PFGE. In addition to promoting good hygiene practices, the implementation of an HACCP plan is necessary to meet the zero-tolerance criteria for Salmonella on beef., (Copyright © 2022 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Genomic DNA preparation is a critical step for successful fingerprinting analysis by Pulsed Field Gel Electrophoresis (PFGE). This paper presents a simple and rapid protocol to prepare DNA samples from up to 24 bacterial isolates simultaneously. It involves performing the conventional PFGE sample preparation steps (cell growth and harvest, agarose-immobilization of intact cells, and DNA release and purification) into a 24-wells culture plate, without subjecting the biological material to repeated transference of containers. The single-container protocol rendered high quality genomic DNA from E. coli clinical isolates. The DNA yields obtained from samples prepared with the single-container protocol showed no differences to those obtained using the conventional DNA sample preparation for PFGE. This procedure is a cost-effective alternative that provides a larger capacity of analysis to the PFGE technique, which could be advantageous in a context of infectious disease outbreaks for pathogens molecular subtyping., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Genetic profiles of Salmonella Minnesota isolates were analyzed using pulsed-field gel electrophoresis (PFGE). In total, 13 isolates obtained from the broiler industry collected in the states of Minas Gerais (11) and São Paulo (2), as well as five recovered from cases of foodborne infections in humans in the states of Minas Gerais (2), Santa Catarina (1), and Rio Grande do Sul (2), were submitted to PFGE. These 18 S. Minnesota isolates together with other 12 of poultry origin were also subjected to antimicrobial susceptibility testing. The PFGE analysis of 18 strains of S. Minnesota generated a dendrogram that grouped the isolates with 83-90% similarity into four main clusters. Among them, cluster "A" grouped the majority of isolates (13), including two of human origin that showed 90% similarity with a broiler isolate, both recovered in Minas Gerais. The S. Minnesota isolates showed resistance to tetracycline (80%), cefoxitin (80%), ceftazidime (46.7%), nalidixic acid (23.3%), ciprofloxacin (13.3%), and streptomycin (10%). No resistance to gentamicin, chloramphenicol, meropenem, nitrofurantoin, and sulfamethoxazole-trimethoprim was found. Moreover, 23.3% of the evaluated isolates presented multi-resistance profile, all from Minas Gerais. The results highlight the importance of further studies involving S. Minnesota, which is prevalent in the Brazilian broiler flocks and could provoke foodborne infection in humans., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

DNA double-strand breaks (DSBs) are highly deleterious lesions, responsible for mutagenesis, chromosomal translocation or cell death. DSB repair (DSBR) is therefore a critical part of the DNA damage response (DDR) to restore molecular and genomic integrity. In humans, this process is achieved through different pathways with various outcomes. The balance between DSB repair activities varies depending on cell types, tissues or individuals. Over the years, several methods have been developed to study variations in DSBR capacity. Here, we mainly focus on functional techniques, which provide dynamic information regarding global DSB repair proficiency or the activity of specific pathways. These methods rely on two kinds of approaches. Indirect techniques, such as pulse field gel electrophoresis (PFGE), the comet assay and immunofluorescence (IF), measure DSB repair capacity by quantifying the time-dependent decrease in DSB levels after exposure to a DNA-damaging agent. On the other hand, cell-free assays and reporter-based methods directly track the repair of an artificial DNA substrate. Each approach has intrinsic advantages and limitations and despite considerable efforts, there is currently no ideal method to quantify DSBR capacity. All techniques provide different information and can be regarded as complementary, but some studies report conflicting results. Parameters such as the type of biological material, the required equipment or the cost of analysis may also limit available options. Improving currently available methods measuring DSBR capacity would be a major step forward and we present direct applications in mechanistic studies, drug development, human biomonitoring and personalized medicine, where DSBR analysis may improve the identification of patients eligible for chemo- and radiotherapy., (Copyright © 2021. Published by Elsevier B.V.)

The aim of the study was to investigate the influence of a pulsed electric field (PEF) on the level of iron ion accumulation in Saccharomyces cerevisiae cells and to select PEF conditions optimal for the highest uptake of this element. Iron ions were accumulated most efficiently when their source was iron (III) nitrate. When the following conditions of PEF treatment were used: voltage 1500 V, pulse width 10 μs, treatment time 20 min, and a number of pulses 1200, accumulation of iron ions in the cells from a 20 h-culture reached a maximum value of 48.01 mg/g dry mass. Application of the optimal PEF conditions thus increased iron accumulation in cells by 157% as compared to the sample enriched with iron without PEF. The second derivative of the FTIR spectra of iron-loaded and -unloaded yeast cells allowed us to determine the functional groups which may be involved in metal ion binding. The exposure of cells to PEF treatment only slightly influenced the biomass and cell viability. However, iron-enriched yeast (both with or without PEF) showed lower fermentative activity than a control sample. Thus obtained yeast biomass containing a high amount of incorporated iron may serve as an alternative to pharmacological supplementation in the state of iron deficiency.

Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.

The ribosomal RNA (rDNA) sequence is the most abundant repetitive element in the budding yeast genome and forms a tandem cluster of ~100-200 copies. Cells frequently change their rDNA copy number, making rDNA the most unstable region in the budding yeast genome. The rDNA region experiences programmed replication fork arrest and subsequent formation of DNA double-strand breaks (DSBs), which are the main drivers of rDNA instability. The rDNA region offers a unique system to understand the mechanisms that respond to replication fork arrest as well as the mechanisms that regulate repeat instability. This chapter describes three methods to assess rDNA instability.

Salmonella is recognized as a major human foodborne pathogen and threat to public health world widely. It is important to carry out epidemiological investigations to determine the primary sources of bacterial contamination. Pulsed-field gel electrophoresis (PFGE) is an important method of the molecular typing, and play an important role in tracking the sources of infection and epidemic control. The PFGE is currently considered as "gold standard" of molecular typing methods for bacterial foodborne pathogen. Here, we describe the PFGE protocol to type the Salmonella from pork.

PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.

The ability of Staphylococcus epidermidis to produce virulence factors, such as biofilm, added to its increased resistance to antimicrobials can cause infections that are difficult to treat. Many staphylococcal virulence factors are under the control of the accessory gene regulator ( agr ). The objective of this study was to establish the agr locus and susceptibility of biofilm-producing S. epidermidis specimens to antimicrobial agents, through PCR reactions, reverse transcription polymerase chain reaction (RT-PCR), and the determination of minimum inhibitory concentration (MIC), and to analyze the clonal profile of 300 strains isolated from blood culture specimens from inpatients at a University Hospital in Brazil, over a 20-year period by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques. The ica operon expression was shown in 83.6% strains, bhp gene in 11.5%, and aap gene in 32.8%. Oxacillin resistance was detected in 90.1%, while 4.9% showed tigecycline resistance, and intermediate resistance to quinupristin/dalfopristin was identified in 0.4%. Clonal profile determination showed 11 clusters, with the ST2 type determined as the major cluster. The S. epidermidis biofilm producer demonstrated a predominance of agr I locus, oxacillin resistance, and SCC mec III as well as the potential dissemination of pathogenic clones in hospital settings over long periods.

Salmonella enterica subspecies enterica causes salmonellosis in humans and animals and is an important cause of food infections worldwide. In recent years, the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA), a fast molecular typing method with strong epidemiological discrimination, has facilitated the effective control of diverse infections. This study aimed at the typing of 28 human origined Salmonella enteritidis, Salmonella infantis, and Salmonella typhimurium strains by using a single MLVA protocol. Previously these strains have been identified by pulsed field gel electrophoresis (PFGE) method and it has been shown that each strain produced a distinct PFGE banding profile. One MLVA protocol was tested on 3 serotypes simultaneously and it produced three banding patterns specific to each of the three common Salmonella serotypes. MLVA also constitute a relatively more cost-effective and faster method than PFGE.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections are a growing concern for public health. The number of sporadic cases and outbreaks of non-O157 STEC infections have increased in recent years. Molecular subtyping is an essential tool that allows high-resolution and rapid differentiation of isolates, identification of case clusters, and detection of outbreak clusters. Multiple-locus variable-number tandem repeat analysis (MLVA) is one of the most useful typing methods for differentiating isolates that cause foodborne diseases. In Japan, serogroups O26, O111, O103, O121, O145, O165, and O91 have been frequently isolated or associated with severe cases of non-O157 STEC infections. In this study, we designed an MLVA scheme (MLVA43) for serogroups O103, O121, O145, O165, and O91 by adding 26 new loci to an MLVA scheme (MLVA17) previously developed by our group for serogroups O157, O26, and O111 using 17 loci. We found that the discriminatory power of MLVA43 was comparable to that of pulsed-field gel electrophoresis (PFGE) for serogroups O103, O145, O165, and O91, and superior to that of PFGE for O121. MLVA43 identified more profiles than did MLVA17, except for serogroup O111 with 707 isolates. The MLVA43 scheme will enable rapid detection of outbreak clusters, which will aid in implementing rapid control measures against non-O157 STEC infections.

Myroides spp. are low-grade opportunistic pathogens. Outbreaks due to Myroides spp. have rarely been described in the literature to date. We report a healthcare-associated outbreak of urinary tract infections (UTIs), caused by Myroides odoratimimus, in a Turkish hospital. As of March 2019 until May 2019, 6 strains of M. odoratimimus were isolated from the urine samples of patients, all of whom were hospitalized in intensive care units. After identification and antibiotic susceptibility testing using the VITEK 2 system, MALDI-TOF-MS and 16S rRNA-based sequencing methods were performed for confirmation and species-level identification. Pulsed-field gel electrophoresis (PFGE) was performed in order to investigate the clonal relatedness of the isolates. All the patients were immunocompromised and underwent urinary catheterization. None of the patients had urinary neoplasm, surgery, or calculi. VITEK 2 and MALDI-TOF-MS systems revealed that the isolates belonged to the Myroides genus; however, the aforementioned systems neglected to identify the isolates at the species level. The isolates were all successfully identified as M. odoratimimus through 16S rRNA-based sequencing. The isolates were resistant to every antibiotic tested. All isolates had an indistinguishable PFGE pattern, thus indicating cross-transmission between cases. Although M. odoratimimus is rarely isolated from human specimens, clinicians should be aware of its ability to cause UTIs and infectious outbreaks.

Introduction: Currently, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis., Materials and Methods: This study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched., Results: All strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2-4 biomarker peaks for the detection of the five PFGE groups and 1-12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively., Conclusion: MALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This protocol describes methods for isolation of total DNA from a strain of Sacchromyces cerevisiae carrying a recombinant yeast artificial chromosome (YAC). This method is appropriate for preparing DNA that will be subjected to regular agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, polymerase chain reaction (PCR), or other methods that do not require intact high-molecular-weight DNA. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. Drop dialysis should be used to exchange buffers. The expected yield from a 10-mL culture is 2-4 µg of yeast DNA., (© 2020 Cold Spring Harbor Laboratory Press.)

Aim: To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model., Methods: We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline., Findings: PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates without evident epidemiological links grouped by PFGE were separated by cgMLST (16/65 isolates) differentiating cities, suggesting that PFGE should be kept for the investigation of local outbreaks. Importantly, hypermutator isolates still shared the pulsotype with their parents (16/65 isolates), whereas they were not recognized by cgMLST. This shows that PFGE was less affected than WGS-based typing by the accelerated genetic drift that occurs in epidemic P. aeruginosa., Conclusions: although WGS-based typing has logically become the new reference standard, we show here that the PFGE can be used with confidence for the investigation of local outbreaks caused by P. aeruginosa., (Copyright © 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Neisseria meningitidis is a significant cause of morbidity and mortality worldwide. Meningococcal isolates have a highly dynamic population structure and can be phenotypically and genetically differentiated into serogroups and clonal complexes. The aim of this study was to describe the phenotypic and genotypic characteristics of invasive isolates recovered in Colombia from 2013 to 2016., Methodology: A total of 193 invasive isolates were analyzed. Phenotypic and genotypic characteristics were determined by serotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing., Results: Based on the results, meningococcal serogroups C, B and Y were responsible for 47.9%, 41.7%, and 9.4% of cases, respectively, and the distribution of serogroups B and C changed over time. Fifteen clonal groups and 14 clonal complexes (cc) were identified by PFGE and genome sequencing. The main clonal group included serogroup B isolates with sequence type (ST)-9493 and its four single-locus variants, which has only been identified in Colombian isolates. The clonal population structure demonstrates that the isolates in this study mainly belong to four clonal complexes: ST-11 cc, ST-32 cc, ST-35 cc and ST-41/44 cc. Thirty-eight penA alleles were identified, but no correlation between MICs and specific sequences was observed., Conclusion: This study shows that most meningococcal isolates recovered from patients with invasive meningococcal disease in Colombia are strains associated with distinct globally disseminated hyperinvasive clones., Competing Interests: The authors have declared that no competing interests exist.

This study compared the ability of pulsed-field gel electrophoresis (PFGE), flaA small variable region (SVR) sequencing, analysis of the clustered regularly interspaced short palindromic repeats locus by high resolution melting analysis (CRISPR-HRMA), and multilocus sequence typing (MLST) for typing 111 Campylobacter jejuni strains isolated from diverse sources during 20 years in Brazil. For this, we used previous results obtained by PFGE and flaA-SVR sequencing from our research group and performed CRISPR-HRMA and MLST typing for the first time. Furthermore, the discrimination index (DI) of each method was accessed. The DI for PFGE, flaA-SVR sequencing, CRISPR-HRMA, and MLST was 0.980, 0.932, 0.868, and 0.931, respectively. By PFGE and flaA-SVR sequencing, some strains from clinical and non-clinical sources and from humans and animals presented ≥ 80% similarity. Similarly, some strains from different origins presented the same ST and CRISPR-HRMA types. In conclusion, despite the different DI values, all assays provided the same epidemiological information suggesting that a potential transmission may have occurred between C. jejuni from clinical and non-clinical sources and from animals and humans in Brazil. Furthermore it was demonstrated the suitability of PFGE that should be used preferably together with MLST and/or flaA-SVR sequencing for typing C. jejuni strains.

Introduction: Knowledge of within-patient dynamics of resistance plasmids during outbreaks is important for understanding the persistence and transmission of plasmid-mediated antimicrobial resistance. During an outbreak of a Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae, the plasmid and chromosomal dynamics of K. pneumoniae within-patients were investigated., Methods: During the outbreak, all K. pneumoniae isolates of colonized or infected patients were collected, regardless of their susceptibility pattern. A selection of isolates was short-read and long-read sequenced. A hybrid assembly of the short-and long-read sequence data was performed. Plasmid contigs were extracted from the hybrid assembly, annotated, and within patient plasmid comparisons were performed., Results: Fifteen K. pneumoniae isolates of six patients were short-read whole-genome sequenced. Whole-genome multi-locus sequence typing revealed a maximum of 4 allele differences between the sequenced isolates. Within patients 1 and 2 the resistance gene- and plasmid replicon-content did differ between the isolates sequenced. Long-read sequencing and hybrid assembly of 4 isolates revealed loss of the entire KPC-gene containing plasmid in the isolates of patient 2 and a recombination event between the plasmids in the isolates of patient 1. This resulted in two different KPC-gene containing plasmids being simultaneously present during the outbreak., Conclusion: During a hospital outbreak of a KPC-producing K. pneumoniae isolate, plasmid loss of the KPC-gene carrying plasmid and plasmid recombination was detected within the isolates from two patients. When investigating outbreaks, one should be aware that plasmid transmission can occur and the possibility of within- and between-patient plasmid variation needs to be considered., Competing Interests: “JR consults for IDbyDNA but that this does not alter our adherence to PLOS ONE policies on sharing data and materials”.

In this work we compared the most frequently used Klebsiella pneumoniae typing methods: PFGE, cgMLST and coreSNP. We evaluated the discriminatory power of the three methods to confirm or exclude nosocomial transmission on K. pneumoniae strains isolated from January to December 2017, in the framework of the routine surveillance for multidrug-resistant organisms at the San Raffaele Hospital, in Milan. We compared the results of the different methods to the results of epidemiological investigation. Our results showed that cgMLST and coreSNP are more discriminant than PFGE, and that both approaches are suitable for transmission analyses. cgMLST appeared to be inferior to coreSNP in the K. pneumoniae CG258 phylogenetic reconstruction. Indeed, we found that the phylogenetic reconstruction based on cgMLST genes wrongly clustered ST258 clade1 and clade2 strains, conversely properly assigned by coreSNP approach. In conclusion, this study provides evidences supporting the reliability of both cgMLST and coreSNP for hospital surveillance programs and highlights the limits of cgMLST scheme genes for phylogenetic reconstructions.

Alteromonas is a widely distributed genus of marine Gammaproteobacteria , with representatives shown to be key players in diverse processes, including biogeochemical cycling and biofouling of marine substrata. While Alteromonas spp. are early colonizers of copper-based antifouling paints on marine vessels, their mechanism of tolerance is poorly understood. PacBio whole-genome sequencing of Alteromonas macleodii strains CUKW and KCC02, isolated from Cu/Ni alloy test coupons submerged in oligotrophic coastal waters, indicated the presence of multiple megaplasmids (ca. 200 kb) in both. A pulsed-field gel electrophoresis method was developed and used to confirm the presence of multiple megaplasmids in these two strains; it was then used to screen additional Alteromonas strains for which little to no sequencing data exist. Plasmids were not detected in any of the other strains. Bioinformatic analysis of the CUKW and KCC02 plasmids identified numerous genes associated with metal resistance. Copper resistance orthologs from both the Escherichia coli Cue and Cus and Pseudomonas syringae Cop systems were present, at times as multiple copies. Metal growth assays in the presence of copper, cobalt, manganese, and zinc performed with 10 Alteromonas strains demonstrated the ability of CUKW and KCC02 to grow at metal concentrations inhibitory to all the other strains tested. This study reports multiple megaplasmids in Alteromonas strains. Bioinformatic analysis of the CUKW and KCC02 plasmids indicate that they harbor elements of the Tra system conjugation apparatus, although their type of mobility remains to be experimentally verified. IMPORTANCE Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early colonizers of copper-based antifouling paint, but their mechanism of tolerance is poorly understood. Sequencing of A. macleodii strains isolated from copper test materials for marine ships indicated the presence of multiple megaplasmids. Plasmids serve as key vectors in horizontal gene transfer and confer traits such as metal resistance, detoxification, ecological interaction, and antibiotic resistance. Bioinformatic analysis identified many metal resistance genes and genes associated with mobility. Understanding the molecular mechanisms and capacity for gene transfer within marine biofilms provides a platform for the development of novel antifouling solutions targeting genes involved in copper tolerance and biofilm formation., (Copyright © 2020 American Society for Microbiology.)

Plasmids are important in carrying antibiotic resistance and other genes between bacterial cells, and a number of methods can be employed to characterize plasmids from clinical isolates. Single colonies typically obtained as part of hospital workflow can undergo S1 nuclease treatment to linearize plasmids followed by pulsed-field gel electrophoresis to enable determination of the number and sizes of plasmids present. Hybridization of S1/PFGE gels can be used to associate replicon types and passenger genes, such as those conferring antibiotic resistance, with a particular plasmid band. Individual plasmids, obtained by conjugation or transformation, can be compared by gel electrophoresis following restriction digestion of plasmid DNA prepared by alkaline lysis methods, including using specialized kits.

Background: Pseudomonas aeruginosa remains one of the most common nosocomial pathogens in intensive care units (ICUs). Although exogenous acquisition has been widely documented in outbreaks, its importance is unclear in non-epidemic situations., Aim: To elucidate the role of exogenous origin of P. aeruginosa in ICU patients., Methods: A chronological analysis of the acquisition of P. aeruginosa was performed using samples collected in 2009 in the DYNAPYO cohort study, during which patients and tap water were screened weekly. Molecular relatedness of P. aeruginosa isolates was investigated by pulsed-field gel electrophoresis. Exogenous acquisition was defined as identification of a P. aeruginosa pulsotype previously isolated from another patient or tap water in the ICU., Findings: The DYNAPYO cohort included 1808 patients (10,402 samples) and 233 water taps (4946 samples). Typing of 1515 isolates from 373 patients and 375 isolates from 81 tap water samples identified 296 pulsotypes. Analysis showed exogenous acquisition in 170 (45.6%) of 373 patients. The pulsotype identified had previously been isolated from another patient and from a tap water sample for 86 and 29 patients, respectively. The results differed according to the ICU., Conclusion: Exogenous acquisition of P. aeruginosa could be prevented in half of patients. The overall findings of this survey support the need for studies on routes of transmission and risk assessment approach to better define how to control exogenous acquisition in ICUs., (Copyright © 2019 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Neonatal sepsis remains a leading cause of neonatal mortality. Maternal bacterial colonization plays a major role in transmission to the infant, with potential for subsequent development of neonatal sepsis with maternally derived strains., Aim: To review the molecular evidence supporting transmission of multidrug-resistant Gram-negative bacteria (MDR-GNB) from colonized mothers to their infants and the risk factors for MDR-GNB transmission., Methods: PubMed and Scopus were searched for studies investigating the mechanisms, risk factors for and/or scale of transmission of MDR-GNB from colonized mothers to their infants. Random effects meta-analyses were performed to determine pooled proportions of MDR-GNB transmission and the neonatal outcomes of transmission., Findings: Eight studies were included in the narrative description and six in the meta-analysis. Five studies used pulsed-field gel electrophoresis to assess relatedness of isolates from colonized mothers and their infants. Pooled proportion of MDR-GNB transmission from colonized mothers to their infants was 27% (95% confidence interval (CI): 8-47%). Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae were the most frequently studied MDR-GNB pathogens transmitted between mother-infant pairs. Following mother-to-infant transmission of an MDR-GNB pathogen, the pooled proportion for the outcome of neonatal colonization was 19% (95% CI: 3-35%)., Conclusion: This systematic review strongly supports MDR and/or ESBL Enterobacteriaceae transmission from colonized mothers to their infants, with subsequent infant colonization. The risk factors contributing to transmission of MDR-GNB between colonized mothers and their infants warrants further research., (Copyright © 2019 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Escherichia coli (E. coli) causes urinary tract infections, pneumonia, surgical site infections, and bloodstream infections and is the important pathogen for both community-acquired and healthcare-associated infections. To investigate the clonality of E. coli is important for infection control and prevention. We aimed to investigate the clonality of clinical E. coli isolates using Cica Geneus E. coli polymerase chain reaction (PCR)-based open-reading frame typing (POT) KIT and clarify the clinical usefulness of this kit. About 124 E. coli isolates obtained from inpatients at Sapporo Medical University Hospital were used. The POT method was used to classify 124 clinical isolates into 87 POT numbers. In addition to the clonality, it was possible to obtain additional information that 20 of the 124 isolates were extended-spectrum β-lactamase (ESBL) producing E. coli (5 isolates of CTX-M-1 group and 15 isolates of CTX-M-9 group) and 13 were sequence type (ST) 131 clone. Furthermore, when these ESBL-producing 20 isolates were compared with pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST), Simpson's index of diversity was 0.968 in POT method, 0.979 in PFGE, and 0.584 in MLST. POT method had an analytical power similar to that of PFGE. In conclusion, attention should be paid to the difference in the interpretation of the results between the POT method and the PFGE, but POT method may be useful to timely monitor the spread of E. coli in medical facilities., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction. PFGE is the 'gold standard' method for bacterial subtyping. However, many strains are non-typable by this approach because of DNA degradation by nucleases action. Aim. To evaluate a modified PFGE protocol for typing nosocomial isolates of Klebsiella pneumoniae . Methods. Twenty- five K. pneumoniae isolates previously exposed to DNA degradation were used to optimize an extraction method for elimination of DNases activity before applying Xba1 enzyme. Introducing of sodium dodecyl sulfate (SDS) in different concentrations to the extraction buffer was evaluated for protecting genomic DNA molecule from degradation by nucleases. Results. Addition of 3 % SDS in combination with 3 % N-lauryl sarcosine to the extraction buffer was found to reduce the previously experienced nuclease activity. Pre-examination of plug quality prior to the digestion phase could efficiently reduce the expense of the wasted enzyme. Conclusion. We have successfully devised a PFGE protocol that enhanced the typeability of nosocomial K. pneumoniae .

We previously reported the distribution of Achromobacter spp. (species and Sequence Types (ST)) in our French Cystic Fibrosis (CF) centre. In the present study we collected 109 Achromobacter isolates (1/patient) from 9 other French CF Centres for species identification, antimicrobial susceptibility testings and Multilocus-Sequence-Typing (MLST) analysis. Ten species were detected, A. xylosoxidans being the most predominant one (73.4% of the isolates). Piperacillin-tazobactam, ceftazidime, imipenem, meropenem and ciprofloxacin were respectively active against 88, 70, 79, 72 and 23% of the isolates. Among the 79 A. xylosoxidans isolates, 46 STs were detected. Interestingly, ST 137, recovered in 4 centres (5 patients), was previously detected in our centre (2 patients). The strains from the 7 patients belonged to the same pulsotype (pulsed-field-gel-electrophoresis analysis) and harboured acquired resistance to meropenem, ceftazidime, ciprofloxacin, and except for 2 isolates, to imipenem and piperacillin-tazobactam. This is the first description in France of a circulating multiresistant A. xylosoxidans strain., (Copyright © 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Pulsed-field gel electrophoresis (PFGE) is the preferred method to measure the size of an insert in a high-capacity vector. After digestion with a restriction enzyme (e.g., PI-SceI or NotI), the DNA fragments are separated by PFGE through a 1% agarose gel., (© 2019 Cold Spring Harbor Laboratory Press.)

Food-producing animals are recognized to play a role in the epidemiology of antimicrobial resistance in Canada. However, the presence of resistant organisms in particular groups of animals, such as chickens raised in small-holder flocks, has not been studied. The purpose of this study was, therefore, to identify and characterize Escherichia coli possessing broad-spectrum β-lactamase genes among a collection of third-generation cephalosporin-resistant isolates recovered from 205 small flocks in southern Ontario. Extended-spectrum β-lactamase (ESBL; CTX-M-1) positive strains were isolated from 26 out of 205 flocks (12.7%), whereas 39 strains possessing AmpC (CMY-2) were grown from 31 out of 205 flocks (15.1%). Pulsed-field gel electrophoresis (PFGE) revealed that the isolates were genetically heterogeneous. Further testing by multi-locus sequence typing confirmed that none of the PFGE-defined clusters belonged to ST131. Our results suggest that the dissemination of this resistance in bacteria isolated from chickens in small-holder flocks may be associated with the spread of plasmids rather than particular E. coli clones and that these isolates do not possess the ESBL types most commonly associated with human infections (CTX-M-15).

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Municipal wastewater treatment plants (WWTPs) may serve as a reservoir for potentially pathogenic and antibiotic resistant bacteria. The discharge of improperly treated wastewater effluent may lead to the spread of these bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) which is responsible for causing pneumonia, septicaemia and skin and soft tissue infections, into the receiving surface waters. This study aimed to determine the antibiogram and virulence gene profiles of MRSA isolates recovered from treated wastewater effluent and receiving surface waters. Genetic fingerprinting of the isolates was also carried out to determine the phylogenetic relationship between the isolates and selected antibiogram profiles. Eighty MRSA isolates were obtained from treated effluent and receiving rivers of two WWTPs in Durban, KwaZulu-Natal. Antibiotic resistance was observed towards lincomycin (100%), oxacillin (98.75%), cefoxitin and penicillin (97.50%), and ampicillin (96.25%). In addition, 72.50%, 66.25%, 52.50%, 40% and 33.75% of isolates showed resistance against cefozolin, azithromycin, amoxicillin/clavulanic acid, erythromycin and vancomycin, respectively. Antibiotic resistance genes detected in the isolates tested in this study: aac(6')/aph(2″) (56.25%), ermC (62.50%), msrA (22.50%), and blaZ and tetK (70%). The frequency of virulence genes: hla and sea was 57.50%, hld was 1.25%, while lukS P/V was 0%. Pulse Field Gel Electrophoresis analysis generated 13 pulsotypes (designated A-M) showing a correlation to their respective antibiograms. Findings from this study showed the presence of potentially pathogenic, multi-drug resistant MRSA in the treated effluent and receiving surface waters. This may have detrimental effects on the health of individuals who come into contact with these water resources.

Pulsed Field Gel Electrophoresis (PFGE) has been considered for many years the 'gold-standard' for characterizing many pathogenic organisms as well as for subtyping bacterial species causing infection outbreaks. This article reviews the basic principles of PFGE and it includes the main advantages and limitations of the different electrode configurations that have been used in PFGE equipment and their influence on the DNA electrophoretic separation. Remarkably, we summarize here the most relevant theoretical and practical aspects that we have learned for more than 20 years developing and using the miniaturized PFGE systems. We also discussed the theoretical aspects related to DNA migration in PFGE agarose gels. It served as the basis for simulating the DNA electrophoretic patterns in CHEF mini gels and mini-chambers during experimental design and optimization. A critical comparison between standard and miniaturized PFGE systems, as well as the enzymatic and non-enzymatic methods for intact immobilized DNA preparation, is provided throughout the review. The PFGE current applications, advantages, limitations and future challenges of the methodology are also discussed., (© 2019 Published by Elsevier Inc.)

We presented a sepsis outbreak caused by Serratia marcescens from contaminated propofol to raise awareness. Three patients had sepsis syndrome after chest surgery. Isolation of S marcescens from patients' respiratory and blood samples alerted us to a possible outbreak. Four syringes filled with propofol and 1 saline solution yielded S marcescens. Nine of 10 isolates from samples of patients and environment genotyped by pulsed-field gel electrophoresis were the same. Disobeying aseptic injection rules of propofol is still causing outbreaks., (Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Several reports describe antimicrobial-resistance transfer among children and the community in outbreak situations, but transfer between a child and a caregiver has not been examined in child care facilities under normal circumstances. We investigated the transfer of antimicrobialresistance genes, resistant bacteria, or both among healthy children and teachers. From 2007 to 2009, 104 Escherichia coli isolates were obtained from four teachers and 38 children in a child care center. Twenty-six cephem-resistant isolates were obtained from children in 2007 and 2008. In 2009, cephem-resistant isolates were detected in children as well as a teacher. Nalidixic acid-resistant isolates from the same teacher for 3 years showed low similarity (<50%) to each other. However, an isolate from a teacher in 2007 and another from a child in 2008 showed high similarity (87%). Pulsed-field gel electrophoresis revealed 100% similarity for four isolates in 2007 and one isolate in 2008, and also similarity among seven isolates carrying the virulence gene (CNF1). This study yielded the following findings: (1) a gene for extended-spectrum β-lactamase was transferred from a child to other children and a teacher; (2) a nalidixic acid-resistant isolate was transferred from a teacher to a child; and (3) a virulent bacterium was transferred between children.

We developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the E. coli reference (ECOR) collection ( n  = 72), O1:K1 isolates causing neonatal meningitis ( n  = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone ( n  = 38), Shiga toxin-producing E. coli (STEC) isolates of serogroups O157:H7 ( n  = 21) and O26 ( n  = 16, 8 of which belonged to an outbreak), 27 Shigella isolates (22 Shigella sonnei isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 Shigella isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of E. coli / Shigella isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE. IMPORTANCE Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all E. coli isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates)., (Copyright © 2019 American Society for Microbiology.)

Background: To analyze the molecular epidemiology and to compare between the major methicillin resistant Staphylococcus aureus biotypes for association with patient characteristics who had an implant for closed fracture and developed early post-operative wound infections (POWI) in a tertiary care hospital of India., Methods: Pulsed-field gel electrophoresis (PFGE), antimicrobial resistance, accessory gene regulator (agr) and staphylococcal cassette chromosome mec (SCCmec) types, Paton-Valentine leukocidin (PVL) gene, toxin gene profiling, biofilm formation and patient demographics were correlated with MLST clonal complexes (CC)., Findings: Overall eight different sequence types (STs) were detected with a predominance of ST239 (66%), ST22 (18%) and some minor types ST772, ST30 (4% each) ST1, ST642, ST6, ST107 (2% each). All ST239 isolates belong to CC239 and SCCmec III whereas ST22 isolates belong to CC22 and SCCmec IV. The isolates varied in the distribution of various toxin genes. With 63.63% biofilm formers ST239 were all multidrug resistant with frequent resistance to erythromycin, clindamycin, gentamicin, cefuroxime, amoxyclav and ciprofloxacin indicating doxycycline, amikacin, vancomycin and linezolid can be the drug of choice., Conclusion: This study shows that ST239 MRSA is still most prevalent strain with new emergence of ST642 and ST107 isolates in association with orthopedic implant based POWI. As compare to other ST types ST239 strain was associated with adverse treatment outcomes. This highlights the importance of improving nosocomial infection control measures in this unit.

This study was conducted to perform an epidemiological survey of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates for nosocomial pneumonia (NP) and visit their Gram-negative bacteria neighbors (GNNs) from the respiratory tracts. Pulsed-field gel electrophoresis and multi locus sequence typing were performed to evaluate the clonal relationships of these isolates. Statistical methods were then used to understand their possible relationship between CRKP and their GNNs. Among the 63 CRKP isolates, 84.1% produced a KPC-2 carbapenemase gene, followed by blaNDM-1 and blaIMP-38 like. 13 different STs and 29 clones were identified. Less heterogeneous clone backgrounds were observed in 53 KPC-2 K. pneumoniae isolates, and 6 STs have been found to contain KPC-2, of which the predominant ST11 harbored 47 KPC-2-producing K. pneumoniae isolates. Particularly, the two infrequent isolates co-possessing blaKPC-2 and blaIMP-38 like shared a different clonal relationship. 87 GNNs isolates of CRKP were established, the analysis present that different strains showed significant or no difference with CRKP upon antibiotics susceptibility. Conclusion, ST11 harboring KPC-2-producing K. pneumoniae isolates were dominant for NP, and both clonal spread and horizontal transfer contributed to the dissemination of CRKP. However, additional studies are needed to explore the biological relationship with their GNNs.

Salmonella enterica serovar Agona strains isolated from human cases were compared to strains that were derived from a clone caused a serovar shift in broilers. Pulsed field gel electrophoresis (PFGE) analysis with XbaI or BlnI digestion showed that three of seven strains from human case strains and most of the 81 strains from broilers were clustered in single complex in a minimum spanning tree (MST) reconstructed from the PFGE data. All the strains from human cases and 22 randomly selected strains from broilers were also analyzed by whole genome sequencing (WGS). Analysis of single nucleotide polymorphism (SNP) in the S. Agona core genes showed that four strains from human cases and all the strains from broilers were clustered in a maximum likelihood phylogenetic tree (ML tree) and an MST. These results indicated that the strains derived from the clone caused the serovar shift had already spread to humans. PFGE analysis with XbaI showed that four strains from broilers did not cluster with the other strains in an MST, though all those strains clustered in an ML tree and an MST reconstructed from SNP data. Moreover, three strains from broilers did not cluster in an MST reconstructed from PFGE with BlnI digestion, though those strains clustered in an ML tree and an MST reconstructed from SNP data. Therefore, it was suggested that S. Agona strains derived from a particular clone could not be traced by PFGE analysis but can be investigated by WGS analysis., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

DNA is one of the most biologically important targets of exogenous and endogenous toxicants as well as carcinogens. Damage to DNA can be of different types (e.g., DNA adducts, DNA protein cross-links, single-strand breaks, oxidized bases, abasic sites, and double-strand breaks (DSBs)). DSBs are considered the most lethal form of DNA damage for eukaryotic cells, and if left unrepaired or misrepaired, can cause cell death, chromosome instability, and cancer. DSBs can arise in the cells through different sources and can be distinguished as endogenous or exogenous DSBs. Exogenous sources can be chemotherapeutic drugs, irradiation, and environmental chemicals. The endogenous causes of DNA DSBs in the cells are mainly reactive oxygen species and faulty repair of oxidative clustered DNA lesions. Qualitative and quantitative analysis of DNA DSBs is of utmost importance to understand physiologically relevant cellular processes as well as to investigate the genotoxic or clastogenic effects of toxicants. Pulsed-field gel electrophoresis (PFGE) is a widely used method for direct quantification of DNA DSBs. In this method, the cells exposed to DSB-inducing agents are embedded in the agarose blocks and lysed. These agarose blocks containing DNA are then run under multiple electric fields which are at 120° angle, to aid in the movement of large DNA strands. It gives a direct and specific measure of DSBs unlike the foci-based assays. This chapter provides a brief overview of the various commonly used approaches to analyze DNA DSBs and describes the theory, advantages and method of PFGE, for use in cells exposed to DNA DSB inducing agents.

The double-strand break (DSB) is the most cytotoxic type of DNA damage and measurement of DSBs in cells is essential to understand their induction and repair. Pulsed-field gel electrophoresis (PFGE) allows for quantitative measurement of DSBs in a cell population generated by DNA damaging agents. PFGE has the capacity to separate DNA molecules from several hundred base pairs to over six million base pairs. In the method described here, molecules from five hundred thousand to three million base pairs are consolidated into a single band on the gel that is readily analyzed.

Acinetobacter baumannii is considered to be an important nosocomial pathogen responsible for various outbreaks that have resulted in a need for effective epidemiological typing methods. Different typing methods are available for A. baumannii epidemiological studies. Currently, the phenotypic typing methods are not being used and replaced by various molecular methods. In this chapter, two important epidemiological typing methods, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), are discussed.

An outbreak of methicillin-resistant Staphylococcus aureus (MRSA) infections on the skin and soft tissues of experimental macaques in the vivarium of The Rockefeller University, New York, triggered this observational and interventional study. We screened 14 macaques in the colony (samples from head, nares, and rectum) and their housing (40 environmental surfaces) four times in 1 year, for S. aureus colonization or contamination, while implementing enhanced decolonization and decontamination procedures. A total of 114 isolates of S. aureus were recovered and characterized (antibiograms, spa typing, multilocus sequence typing, pulsed-field gel electrophoresis [PFGE], mecA, Panton-Valentine Leukocidin, and arginine catabolic mobile element). Based on these results, six strains of S. aureus were identified: two MRSA strains (t16708/ST3862/PFGE-A, t16709/ST3862/PFGE-C) and one methicillin-sensitive S. aureus (t8397/ST3884/PFGE-D) were characterized for the first time in this study; strains belonging to spa types t189 and t4167 have been identified in primates in previous studies. None of these strains was common to the neighboring New York City human community. Thus, it seems probable that the animals were already colonized upon arrival to the University. We suggest screening primates for S. aureus carriage upon arrival to University vivaria and possible implementation of extensive decolonization procedures before any surgical interventions.

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and nonspecific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein recovery efficiency. Here, we describe the enhancement of protein separation efficiency for up to twofold in conventional one-dimensional PAG electrophoresis (1D PAGE), two-dimensional (2D) PAGE, and native PAGE by implementing pulses of inverted electric field during gel electrophoresis.

Rapid and accurate identification of pathogen is a major quarantine strategy for outbreak prevention. We used capillary electrophoresis-random amplified polymorphic DNA (CE-RAPD) to generate highly discriminatory pathogen profiles, reduced batch effects between profiles by novel normalization procedure and pattern of technical repeats, followed by target similarity evaluation using target identification score (TIS). A full target signature contains several patterns. TIS system was optimized by training set isolates that included three species, and validated using two hundred clinical Klebsiella pneumoniae isolates. Hierarchical clustering analysis showed CE-RAPD profiles arrange clusters according to the species or the source. Moreover, samples with similar profile may display similar antibiotic susceptibility. By using a signature of four patterns, the TIS system could accurately identify target among different isolates. The variation between isolates may be caused by small change in genome. TIS system provides a standardized tool for building of outbreak firewall and facilitate data exchange.

Background: Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases in the world. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing methods were used to characterize Staphylococcus aureus isolates from food surveillance during 2013-2015 in southwest China, and Staphylococcal cassette chromosome mec (SCCmec) typing was used for methicillin-resistant S. aureus (MRSA). Isolates were also examined for their antibiotic resistance and carriage of virulence genes., Results: Isolation rate of S. aureus was 2.60% during the three years' surveillance and 29.50% of them were MRSA. All the S. aureus had hla genes (100%), 14.34% of the strains had tst, and 16.73% had PVL. 163 PFGE-SmaI patterns, 41 ST types and 36 spa types were obtained for all the S. aureus. Among them, ST6-t701 (13.15%), ST7-t091 (12.75%), ST59-t437 (9.96%) and ST5-t002 (7.57%) were the prevalent genotypes. Most of MRSA in this study belonged to SCCmec IV and V, accounted for 74.32% and 20.27% respectively. ST6-SCCmec IV-t701 (36.50%) was the most prevalent clone among isolates from food, followed by ST59-SCCmec V-t437 (20.30%), ST5-SCCmec IV-t002 (12.20%) and ST59-SCCmec IV-t437 (12.20%). Some strains had the identical PFGE patterns, ST and spa types with isolates from patients., Conclusions: S. aureus isolated from food in southwest China displayed heterogeneity. Isolates had the same genotype profiles with isolates from patients, indicating high homology.

Cystic fibrosis (CF) is an inherited recessive disease that affects mucocillary clearance in the lung, allowing it to be colonised with bacteria such as Staphylococcus aureus. To survive in the CF lung S. aureus adapts both phenotypically and genotypically, through various mechanisms. In this study, multiple specimens were collected from the participants and were processed routinely and were additionally cultured in chromogenic media. Multiplex PCR assays were employed to detect methicillin resistance and selected virulence and quaternary ammonium compound (qac) genes. Genetic relatedness of the S. aureus was determined using agr, SCCmec and spa typing as well as pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Thirty-three S. aureus isolates were isolated, of which 51% (17/33) were methicillin resistant S. aureus (MRSA). The virulence and qac genes were more prevalent in MRSA than the methicillin sensitive S. aureus (MSSA) isolates. The PFGE analysis showed nine distinct pulsotypes while MLST showed eight sequence types. All the STs detected in this study, except for ST508 have been previously isolated from CF patients according to the literature. This study showed a genetically diverse S. aureus population with a high prevalence of virulence genes among the MRSA isolates from the CF clinic.