Comparison of CRISPR typing and conventional molecular methods for distinguishing Laribacter hongkongensis isolates from fish, frogs and humans.

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.

Customizable BAC-based DNA markers for pulsed-field gel electrophoresis.

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.

Validating the Utility of Multilocus Variable Number Tandem-repeat Analysis (MLVA) as a Subtyping Strategy to Monitor Listeria monocytogenes In-built Food Processing Environments.

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.

Subtyping of Campylobacter coli isolated from raw poultry meat in retail markets using amplified intergenic locus polymorphism - A novel rapid subtyping method.

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.

Molecular subtyping of Salmonella spp. strains in provincial abattoirs with no hazard analysis critical control point from Buenos Aires, Argentina.

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.

A single-container procedure for multiple genomic DNA samples preparation for pulsed field gel electrophoresis.

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.

Molecular genotyping reveals inter-regional relatedness among antimicrobial resistant Salmonella Minnesota strains isolated from poultry farm and humans, Brazil.

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.

Caracterização molecular de Staphylococcus aureus isolados de queijos artesanais da Serra da Canastra / Molecular characterization of Staphylococcus aureus isolated from artisanal cheeses from Serra da Canastra

O queijo Minas Artesanal da Canastra é produzido na Serra da Canastra (MG), utilizando leite cru, coalho e pingo, que é uma cultura endógena natural de cada queijaria. Devido ao uso de leite cru, o produto pode veicular microrganismos causadores de doenças veiculadas por alimentos, como Staphylococcus aureus. A caracterização molecular é uma ferramenta importante para avaliar a população microbiana do alimento e direcionar a aplicação de medidas de controle na produção. Este estudo caracterizou a diversidade genética, o potencial de virulência e determinou o perfil de susceptibilidade a antimicrobianos de S. aureus isolados de queijos produzidos na Serra da Canastra. Para o estudo transversal foram analisados 248 isolados de queijos que tinham um tempo de maturação de 22 dias, provenientes de 83 propriedades. Por outro lado, no estudo longitudinal foram analisados outros 197 isolados coletados ao longo do processo de maturação, provenientes de três propriedades. Os isolados foram submetidos a testes bioquímicos para confirmação do gênero e para a confirmação da espécie de S. aureus, foi identificado o gene nuc por meio da técnica de PCR. Além disso, foi pesquisado o gene mecA para a detecção de S. aureus Resistente a Meticilina (MRSA). Após os testes de confirmação, 144 isolados do estudo transversal e 159 do estudo longitudinal foram positivos para o gene nuc, específico para S. aureus. Posteriormente, o perfil clonal foi determinado por Eletroforese de Campo Pulsado (PFGE) utilizando a enzima SmaI e tipagem do locus agr por PCR multiplex. A análise por PFGE foi realizada no programa BioNumerics. A técnica PCR foi realizada para identificar a presença de genes que codificam a produção de hemolisinas, toxina TSST-1, enterotoxinas SEs (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEO, SEM), formação de biofilme e Componentes Microbianos de Superfície que Reconhecem a Matriz de Moléculas Adesivas (MSCRAMMs). Os isolados foram submetidos ao teste de susceptibilidade a antimicrobianos por disco de difusão. Por último, a formação de biofilme em microplaca de 96 poços, em caldo TSB a 37°C, foi verificada pela metodologia de Cristal Violeta. O gene mecA foi detectado em 1,9% dos 445 isolados. A tipagem agrrevelou que 83 (27,4%) dos isolados são do tipo agr-I, 95 (31,4%) agr-II e 43 (14,2%) agr-III, sendo que não foram detectados isolados classificados como agr-IV. A tipagem por PFGE revelou um total de 54 perfis. Assim, um isolado representativo de cada perfil foi utilizado nos demais testes que mostraram a presença dos genótipos spa mais frequentes t127 e t605 (20,58%); t002 (14,70%), seguidos pelos tipos t267 (8,82%); t1234 e t693 (5,8%) e t021, t177, t306, t321, t359, t442, t521, t693 e t5493 (2,9%). Além disso, encontramos a presença dos genes do grupo SEs, sea 1 (1,8%), seh 11 (20,3%), sei 10 (18,5%), sej 7 (12,9%), seg e seo 14 (25,9%), sem 8 (14,8%), e os genes seb, sec, sed, see e tst não foram detectados. Para os genes das hemolisinas, hla foi positivo em todos os isolados e hlb foi positivo em 53 (98,1%) isolados. Os genes positivos para MSCRAMMS foram fnbA, fnbB 18 (33,3%), clfA, clfB e eno 53 (98,1%), fib 44 (81,4%), bbp 4 (7,4%), cna 17 (31,4%) e ebps 10 (18,5%). Por último, os genes de formação de biofilme icaA e icaD estiveram presentes em 38 (70,3%) e 25 (46,2%) dos isolados, respectivamente. Na avaliação de susceptibilidade a antibióticos dos 54 isolados escolhidos, 25 (46,3%) apresentaram maior resistência a penicilina e 13 (24,0%) a tetraciclina. Em menor porcentagem (1,8%), 1 isolado cada foi resistente a eritromicina, cefoxitina, clindamicina, gentamicina, cotrimazol, azitromicina e trimetropim. Além disso, 8 isolados (14,8%) apresentaram resistência intermediaria a tetraciclina, 3 (5,5%) a gentamicina e 1 (1,8%) a tobramicina. No teste para a determinação da formação de biofilme por cristal violeta, 13,7%, foram classificados em isolados não formadores, 60,8% em fracamente formadores, 25,5% moderadamente formadores e nenhum como fortemente formador. A alta diversidade de cepas de S. aureus observada neste estudo mostrou que existem vários tipos de linhagens circulando na região da Canastra. A caracterização revelou uma elevada frequência de genes de virulência e que mais estudos são necessários para avaliar o potencial de produção de enterotoxinas nos queijos artesanais. A melhora dos procedimentos de higienização durante todas as etapas de produção pode ser uma solução para a redução dos níveis de contaminação por S. aureus

Canastra Minas Artesanal cheese is produced in Serra da Canastra (MG), using raw milk, rennet and a natural endogenous culture called pingo. Due to the use of raw milk, the product can carry microorganisms that cause foodborne diseases, such as Staphylococcus aureus. Molecular characterization is an important tool to assess the microbial population of food and guide the application of control measures in production. This study characterized the genetic diversity, virulence potential and determined the antimicrobial susceptibility profile of S. aureus isolated from cheeses produced in Serra da Canastra. A total of 248 isolates from 22 days ripened cheeses were obtained from 83 properties (cross sectional study). Another 197 isolates were collected during maturation (longitudinal study), in three properties. The isolates were submitted to biochemical tests to confirm the genus and to confirm the S. aureus species, the nuc gene was identified by PCR. In addition, the detection of mecA gene was performed for the detection of Methicillin Resistant S. aureus (MRSA). After confirmation tests, 144 isolates from the cross-sectional study and 159 from the longitudinal study were positive for the nuc gene, specific for S. aureus. Subsequently, the clonal profile of the isolates was determined by Pulsed Field Gel Electrophoresis (PFGE) using the SmaI enzyme and typing of the agr locus by multiplex PCR. PFGE analysis was performed using the BioNumerics program. PCR was performed to identify the presence of genes encoding the production of hemolysins, TSST-1 toxin, enterotoxins SEs (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEO, SEM), biofilm formation and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The isolates were submitted to the antimicrobial susceptibility test by disc diffusion. Finally, biofilm formation in a 96-well microplate in TSB broth at 37°C was verified by the Cristal Violeta method. The mecA gene was detected in 1.9% of the 445 isolates. Agr typing revealed that 83 (27.4%) of the isolates are agr-I, 95 (31.4%) agr-II and 43 (14.2%) agr-III, and no isolate was classified as agr-IV. PFGE typing revealed a total of 54 profiles. Thus, a representative isolate of each profile was used in the other tests that showed the presence of the most frequent spagenotypes t127, t605 (20.58%); t002 (14.70%), followed by types t267 (8.82%); t1234, t693 (5.8%) e t021, t177, t306, t321, t359, t442, t521, t693 and t5493 (2.9%). In addition, we found the presence of the genes of the SEs group: sea 1 (1.8%), seh 11 (20.3%), sei 10 (18.5%), sej 7 (12.9%), seg and seo 14 (25.9%), sem 8 (14.8%), while seb, sec, sed, see and tst genes were not detected. For hemolysin genes, hla was positive in all isolates and hlb was positive in 53 (98.1%) isolates. The positive genes for MSCRAMMS were: fnbA, fnbB 18 (33.3%), clfA, clfB e eno 53 (98.1%), fib 44 (81.4%), bbp 4 (7.4%), cna 17 (31.4%) and ebps 10 (18.5%). Finally, the biofilm formation genes icaA and icaD were present in 38 (70.3%) and 25 (46.2%) of the isolates, respectively. In the evaluation of antibiotic susceptibility of the 54 isolates, 25 (46.3%) showed greater resistance to penicillin and 13 (24.0%) to tetracycline. In a lower percentage (1.8%), 1 isolate each was resistant to erythromycin, cefoxitin, clindamycin, gentamicin, contrimazole, azithromycin and trimethoprim. In addition, 8 isolates (14.8%) showed intermediate resistance to tetracycline, 3 (5.5%) to gentamicin and 1 (1.8%) to tobramycin. In the test for the determination of biofilm formation by crystal violet, 13.7% were classified as non-forming isolates, 60.8% as weakly forming, 25.5% moderately forming and none as strongly forming. The high diversity of S. aureus strains observed in this study showed that there are several types of strains circulating in the Canastra region. The characterization revealed a high frequency of virulence genes and that further studies are needed to assess the potential for enterotoxin production in artisanal cheeses. The improvement of hygiene procedures during all stages of production can be a solution for reducing the levels of contamination by S. aureus

Evaluation of DNA double-strand break repair capacity in human cells: Critical overview of current functional methods.

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.

Pulsed Electric Field (PEF) Enhances Iron Uptake by the Yeast Saccharomyces cerevisiae.

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.

Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction.

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.

Gel Electrophoresis Analysis of rDNA Instability in Saccharomyces cerevisiae.

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.

Molecular Typing of Salmonella by Pulsed-Field Gel Electrophoresis.

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.

Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Listeria monocytogenes.

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.

Characterization of AmpC Beta-lactamase mutations of extensively drug-resistant Pseudomonas aeruginosa isolates that develop resistance to ceftolozane/tazobactam during therapy / Caracterización de las mutaciones en la betalactamasa AmpC de aislados de Pseudomonas aeruginosa XDR que desarrollaron resistencia a ceftolozano/tazobactam durante el tratamiento

INTRODUCTION: We characterized AmpC β-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. METHODS: Five pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. RESULTS: The ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229-E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. CONCLUSIONS: Ceftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent

INTRODUCCIÓN: Se han caracterizado las mutaciones en la betalactamasa AmpC que han producido resistencia a ceftolozano/tazobactam en aislados de Pseudomonas aeruginosa extremadamente resistente (XDR) en pacientes tratados con este agente desde junio de 2016 hasta diciembre de 2018. MÉTODOS: Se incluyeron 5 pares de aislados (sensibles/resistentes a ceftolozano/tazobactam) de P. aeruginosa XDR entre un total de 49 pacientes tratados. Se estudió la relación clonal mediante electroforesis en campo pulsado y MLST. Las mutaciones en AmpC se caracterizaron mediante amplificación por PCR del gen blaPDC y posterior secuenciación. RESULTADOS: Se detectó el clon de alto riesgo ST175 en 4 pares de aislados y el ST1182 en el restante. Todos los aislados resistentes mostraron una mutación en AmpC: T96I en 2 aislados, E247K, G183V y una deleción de 19 aminoácidos (G229-E247) en los otros 3. La mutación G183V no había sido descrita antes. Los 5 aislados resistentes a ceftolozano/tazobactam mostraron resistencia cruzada a ceftazidima/avibactam y CMI inferiores de imipenem y piperacilina/tazobactam que los aislados sensibles. CONCLUSIONES: La resistencia a ceftolozano/tazobactam se asoció con mutaciones en AmpC en todos los casos, incluida una nueva mutación G183V no descrita con anterioridad. La vigilancia y caracterización de la resistencia emergente a ceftolozano/tazobactam es de gran importancia para preservar este nuevo agente antipseudomónico

Detection of the agr System and Resistance to Antimicrobials in Biofilm-Producing S. epidermidis.

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 SCCmec III as well as the potential dissemination of pathogenic clones in hospital settings over long periods.

A Healthcare-Associated Outbreak of Urinary Tract Infections Due to Myroides odoratimimus.

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.

Molecular Typing of Different Salmonella Serotypes by Multiple-Locus Analysis of Tandem Repeats.

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.

Growth of Saccharomyces cerevisiae and Preparation of DNA.

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.

Quick identification and epidemiological characterization of Francisella tularensis by MALDI-TOF mass spectrometry.

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.