Plasmid-mediated Kluyvera-like arnBCADTEF operon confers colistin (hetero)resistance to Escherichia coli.

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB system and by still unknown mechanisms. This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to strains with mutations that confer colistin resistance. The expression of mcr genes negatively controls the transcription of the arnBCADTEF operon itself, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 62.1 Kb encoding an extra copy of the arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, called pArnT1, was cured by ethidium bromide and the cells lost resistance to colistin in parallel. Furthermore, a susceptible E. coli strain acquired heteroresistance after transformation with pArnT1 or pBAD24 carrying the Kluyvera-like arnBCADTEF operon, revealing it as a new colistin resistance determinant.

Evaluation of the broth microdilution plate methodology for susceptibility testing of Mycobacterium tuberculosis in Peru.

BACKGROUND: Tuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru. METHODS: MTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods. RESULTS: MIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179). CONCLUSIONS: The BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

Listeria monocytogenes survives better at lower storage temperatures in regular and low-salt soft and cured cheeses.

The behaviour of Listeria monocytogenes was investigated in soft pasteurized milk cheese elaborated with different salt concentrations (1.17 and 0.30% w/w) and in cured raw sheep milk cheese over storage up to 189 days at different isothermal conditions. Commercial 25-g cheese samples were inoculated with a 4-strain cocktail of L. monocytogenes (serovars 4b, 1/2a, 1/2b and 1/2c) at approximately 104 CFU/g. The inoculated samples were stored at 4 and 22 °C and withdrawn at proper intervals for L. monocytogenes enumeration. The prevalence of the different serovar strains of L. monocytogenes was characterized on soft cheese samples over storage at 4 °C using multiplex PCR. Salt reduction did not affect the survival of L. monocytogenes in soft cheeses and a maximum of 1-log reduction was observed in both regular and low-salt cheeses after 189 days of storage at 4 °C. The pathogen showed greater survival capacity in both soft and cured cheeses during storage at 4 °C compared to the storage at 22 °C, where more than 2.5 log reductions were computed. The fate of L. monocytogenes was described through a Weibull model fitted to survival data. The time required for a first tenfold reduction of the L. monocytogenes population (δ) at 4 °C is around 150 days in soft and 72 days in cured cheeses. At 22 °C, the estimated δ values are at least 60% lower in both cheese types. Among the four L. monocytogenes serovars present in the inoculated cocktail, the serovar 4b strain was the most sensitive to refrigerated storage, while the prevalence of serovar 1/2c strain increased over time in soft cheeses. Overall, the data obtained in this study help to deepen knowledge into factors affecting L. monocytogenes behaviour on cheeses and evidenced the variability between serovars in terms of survival capacity, which may be considered when performing microbial risk assessments.

TORMES: an automated pipeline for whole bacterial genome analysis.

MOTIVATION: The progress of High Throughput Sequencing (HTS) technologies and the reduction in the sequencing costs are such that Whole Genome Sequencing (WGS) could replace many traditional laboratory assays and procedures. Exploiting the volume of data produced by HTS platforms requires substantial computing skills and this is the main bottleneck in the implementation of WGS as a routine laboratory technique. The way in which the vast amount of results are presented to researchers and clinicians with no specialist knowledge of genome sequencing is also a significant issue. RESULTS: Here we present TORMES, a user-friendly pipeline for WGS analysis of bacteria from any origin generated by HTS on Illumina platforms. TORMES is designed for non-bioinformatician users, and automates the steps required for WGS analysis directly from the raw sequence data: sequence quality filtering, de novo assembly, draft genome ordering against a reference, genome annotation, multi-locus sequence typing (MLST), searching for antibiotic resistance and virulence genes, and pangenome comparisons. Once the analysis is finished, TORMES generates and interactive web-like report that can be opened in any web browser and shared and revised by researchers in a simple manner. TORMES can be run by using very simple commands and represent a quick an easy way to perform WGS analysis. AVAILABILITY AND IMPLEMENTATION: TORMES is free available at https://github.com/nmquijada/tormes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Bioinformatics of next generation sequencing in clinical microbiology diagnosis]. / Aplicación de la secuenciación masiva y la bioinformática al diagnóstico microbiológico clínico.

Massive parallel sequencing (High-Throughput Sequencing [HTS]) allows to read millions or billions of DNA sequences or fragments (reads) in parallel and is revolutionizing microbiology research, moving from laboratory methods to computed-assisted analyses, with the compelling use of Bioinformatics. The time and cost reduction in studies on the microbiota, microbiome and metagenome, allows to rapidly progress in diagnosis, taxonomy, epidemiology, comparative genomics, virulence, discovery of genes or variants of interest and the association of microorganisms with traditionally considered non-microbial diseases. In this review, the terminology, the sequencing technologies and their applications are described for microbial analysis using open-source bioinformatics software, analysis pipelines, databases and web platforms that allow a user-friendly bioinformatics approach affordable by the clinical microbiologist and infectious disease practitioners.

Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo.

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.

Oxacillin-susceptible mecA-positive Staphylococcus aureus associated with processed food in Europe.

We report for the first time an oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with a processed food product in Europe. One isolate (MRSA-ST5-type V SCCmec) was found in cheese among 600 food samples confiscated from air passengers from international flights in Vienna Airport (Austria). Type V SCCmec strains do not harbor functional mecI-mecR1 genes and in such strains mecA expression is regulated by the bla system (blaI-blaR1-blaZ). It has been recently reported that malfunctions in the bla system lead to the constitutive expression of mecA. The OS-MRSA reported in this study harbored the bla system on a plasmid and one deletion occurred in the blaR1 gene causing a frameshift variant that lead to an incomplete BlaR1 protein. This finding highlights the potential role of food as a neglected route of dissemination of emerging MRSA variants.

Different Lactobacillus populations dominate in "Chorizo de León" manufacturing performed in different production plants.

"Chorizo de Léon" is a high-value Spanish dry fermented sausage traditionally manufactured without the use of starter cultures, owing to the activity of a house-specific autochthonous microbiota that naturally contaminates the meat from the environment, the equipment and the raw materials. Lactic acid bacteria (particularly Lactobacillus) and coagulase-negative cocci (mainly Staphylococcus) have been reported as the most important bacterial groups regarding the organoleptic and safety properties of the dry fermented sausages. In this study, samples from raw minced meat to final products were taken from five different producers and the microbial diversity was investigated by high-throughput sequencing of 16S rRNA gene amplicons. The diverse microbial composition observed during the first stages of "Chorizo de Léon" evolved during ripening to a microbiota mainly composed by Lactobacillus in the final product. Oligotyping performed on 16S rRNA gene sequences of Lactobacillus and Staphylococcus populations revealed sub-genus level diversity within the different manufacturers, likely responsible of the characteristic organoleptic properties of the products from different companies.

Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.

Colistin resistance genes mcr-3 and mcr-1 have been detected in an Escherichia coli isolate from cattle faeces in a Spanish slaughterhouse in 2015. The sequences of both genes hybridised to same plasmid band of ca 250 kb, although colistin resistance was non-mobilisable. The isolate was producing extended-spectrum beta-lactamases and belonged to serotype O9:H10 and sequence type ST533. Here we report an mcr-3 gene detected in Europe following earlier reports from Asia and the United States.

Molecular investigation of tularemia outbreaks, Spain, 1997-2008.

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

Methicillin-resistant Staphylococcus aureus harboring mecC in livestock in Spain.

We report for the first time mecC-positive methicillin-resistant Staphylococcus aureus (mecC-MRSA) in livestock in Spain. One isolate (sequence type 130) was found in milk samples among 601 S. aureus isolates obtained from 229 dairy sheep farms. This finding highlights the potential for zoonotic transmission of mecC-positive MRSA and the need for surveillance programs to monitor its presence and clonal evolution.

Whole Genome Sequencing of Mycobacterium tuberculosis under routine conditions in a high-burden area of multidrug-resistant tuberculosis in Peru.

Whole Genome Sequencing (WGS) is a promising tool in the global fight against tuberculosis (TB). The aim of this study was to evaluate the use of WGS in routine conditions for detection of drug resistance markers and transmission clusters in a multidrug-resistant TB hot-spot area in Peru. For this, 140 drug-resistant Mycobacterium tuberculosis strains from Lima and Callao were prospectively selected and processed through routine (GenoType MTBDRsl and BACTEC MGIT) and WGS workflows, simultaneously. Resistance was determined in accordance with the World Health Organization mutation catalogue. Agreements between WGS and BACTEC results were calculated for rifampicin, isoniazid, pyrazinamide, moxifloxacin, levofloxacin, amikacin and capreomycin. Transmission clusters were determined using different cut-off values of Single Nucleotide Polymorphism differences. 100% (140/140) of strains had valid WGS results for 13 anti-TB drugs. However, the availability of final, definitive phenotypic BACTEC MGIT results varied by drug with 10-17% of invalid results for the seven compared drugs. The median time to obtain results of WGS for the complete set of drugs was 11.5 days, compared to 28.6-52.6 days for the routine workflow. Overall categorical agreement by WGS and BACTEC MGIT for the compared drugs was 96.5%. Kappa index was good (0.65≤k≤1.00), except for moxifloxacin, but the sensitivity and specificity values were high for all cases. 97.9% (137/140) of strains were characterized with only one sublineage (134 belonging to "lineage 4" and 3 to "lineage 2"), and 2.1% (3/140) were mixed strains presenting two different sublineages. Clustering rates of 3.6% (5/140), 17.9% (25/140) and 22.1% (31/140) were obtained for 5, 10 and 12 SNP cut-off values, respectively. In conclusion, routine WGS has a high diagnostic accuracy to detect resistance against key current anti-TB drugs, allowing results to be obtained through a single analysis and helping to cut quickly the chain of transmission of drug-resistant TB in Peru.

Real-time PCR in Food Science: Introduction.

Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

Real-time PCR in Food Science: PCR Diagnostics.

A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

Molecular characterization of Mycoplasma agalactiae reveals the presence of an endemic clone in Spain.

Mycoplasma agalactiae isolates from Spain were genetically characterized to investigate their genomic diversity and to better understand their relationship to isolates from other countries. Molecular typing revealed a high genomic homogeneity in Spanish M. agalactiae isolates, which clearly shows the circulation of one endemic clonal population.

TRRAP-mediated acetylation on Sp1 regulates adult neurogenesis.

The adult hippocampal neurogenesis plays a vital role in the function of the central nervous system (CNS), including memory consolidation, cognitive flexibility, emotional function, and social behavior. The deficiency of adult neural stem cells (aNSCs) in maintaining the quiescence and entering cell cycle, self-renewal and differentiation capacity is detrimental to the functional integrity of neurons and cognition of the adult brain. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) have been shown to modulate brain functionality and are important for embryonic neurogenesis via regulation of gene transcription. We showed previously that Trrap, an adapter for several HAT complexes, is required for Sp1 transcriptional control of the microtubule dynamics in neuronal cells. Here, we find that Trrap deletion compromises self-renewal and differentiation of aNSCs in mice and in cultures. We find that the acetylation status of lysine residues K16, K19, K703 and K639 all fail to overcome Trrap-deficiency-incurred instability of Sp1, indicating a scaffold role of Trrap. Interestingly, the deacetylation of Sp1 at K639 and K703 greatly increases Sp1 binding to the promoter of target genes, which antagonizes Trrap binding, and thereby elevates Sp1 activity. However, only deacetylated K639 is refractory to Trrap deficiency and corrects the differentiation defects of Trrap-deleted aNSCs. We demonstrate that the acetylation pattern at K639 by HATs dictates the role of Sp1 in the regulation of adult neurogenesis.

Bacteriophages as Biotechnological Tools.

Bacteriophages are ubiquitous organisms that can be specific to one or multiple strains of hosts, in addition to being the most abundant entities on the planet. It is estimated that they exceed ten times the total number of bacteria. They are classified as temperate, which means that phages can integrate their genome into the host genome, originating a prophage that replicates with the host cell and may confer immunity against infection by the same type of phage; and lytics, those with greater biotechnological interest and are viruses that lyse the host cell at the end of its reproductive cycle. When lysogenic, they are capable of disseminating bacterial antibiotic resistance genes through horizontal gene transfer. When professionally lytic-that is, obligately lytic and not recently descended from a temperate ancestor-they become allies in bacterial control in ecological imbalance scenarios; these viruses have a biofilm-reducing capacity. Phage therapy has also been advocated by the scientific community, given the uniqueness of issues related to the control of microorganisms and biofilm production when compared to other commonly used techniques. The advantages of using bacteriophages appear as a viable and promising alternative. This review will provide updates on the landscape of phage applications for the biocontrol of pathogens in industrial settings and healthcare.

Hepatitis E virus in pork production chain in Czech Republic, Italy, and Spain, 2010.

We evaluated the prevalence of hepatitis E virus (HEV) in the pork production chain in Czech Republic, Italy, and Spain during 2010. A total of 337 fecal, liver, and meat samples from animals at slaughterhouses were tested for HEV by real-time quantitative PCR. Overall, HEV was higher in Italy (53%) and Spain (39%) than in Czech Republic (7.5%). HEV was detected most frequently in feces in Italy (41%) and Spain (39%) and in liver (5%) and meat (2.5%) in Czech Republic. Of 313 sausages sampled at processing and point of sale, HEV was detected only in Spain (6%). HEV sequencing confirmed only g3 HEV strains. Indicator virus (porcine adenovirus) was ubiquitous in fecal samples and absent in liver samples and was detected in 1 slaughterhouse meat sample. At point of sale, we found porcine adenovirus in sausages (1%-2%). The possible dissemination of HEV and other fecal viruses through pork production demands containment measures.

A survey of Mycoplasma agalactiae in dairy sheep farms in Spain.

BACKGROUND: Contagious Agalactia (CA) is one of the major animal health problems in small ruminants because of its economic significance. Currently, four Mycoplasma spp. have been associated with this syndrome: M. agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. Their presence has been evaluated in several studies conducted in CA-endemic countries. However, previous Spanish studies have been focused on caprine CA, and there is a knowledge gap regarding which Mycoplasma species are present in sheep flocks from Spain, which has the second highest number of sheep amongst the 27 European Union member states. Consequently, we investigated the presence and geographic distribution of the four CA-causing mycoplasmas in Spanish dairy sheep farms. This is the first time such an investigation has been performed. RESULTS: Three hundred thirty nine out of 922 sheep flocks were positive for M. agalactiae by real time PCR (36.8%) and 85 by microbiological identification (9.2%). Interestingly, all 597 milk samples assessed for the presence of M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens tested negative. To evaluate the intermittent excretion of the pathogen in milk, we sampled 391 additional farms from 2 to 5 times, resulting that in 26.3% of the cases a previously positive farm tested negative in a later sampling. CONCLUSIONS: M. agalactiae was the only Mycoplasma species detected in the study area showing a high frequency of presence and wide distribution. Therefore, the establishment of a permanent surveillance network is advantageous, as well as the implementation of control and prevention measures to hinder the dissemination of M. agalactiae and to prevent the entrance of other Mycoplasma species.

Enteric viruses in food safety: New threats for an old problem.

Foodborne diseases are one of the most serious concerns in public health. It is estimated that around 600 million cases of gastroenteritis occur worldwide each year. At present, more than 200 food-borne diseases are known, which can cause from mild gastroenteritis to syndromes with a fatal outcome, with the added possibility of chronic complications. One of the major etiological agents in foodborne diseases are the food and waterborne viruses, which are attracting a great deal of attention to researchers, food hygienists and policy makers. Several aspects differentiate these pathogens from foodborne pathogenic bacteria: their high capacity for infection and preservation in food environments, and their difficulty for a correct and sensitive detection. In recent years, different initiatives have been carried out to prioritize research in the area of viruses in food, prioritizing different aspects of their detection, epidemiology and control. There is clear evidence that the existing data on their prevalence may be underestimated due to the lack of robust methods for their sensitive detection. It is also necessary to know exactly what the incidence is in the different stages of the food production chain, and particularly in that which is dedicated to the transformation of products of animal origin. Finally, it is also necessary to calibrate the current disinfection procedures in the food industry in order to reliably establish a quantitative evaluation of the viral risk in food.