Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae

Recent advances in the development and use of molecular tests to predict antimicrobial resistance in Neisseria gonorrhoeae.

INTRODUCTION The number of genetic tests, mostly real-time PCRs, to detect antimicrobial resistance (AMR) determinants and predict AMR in Neisseria gonorrhoeae is increasing. Several of these assays are promising, but there are important shortcomings and few assays have been adequately validated and quality assured. Areas covered: Recent advances, focusing on publications since 2012, in the development and use of molecular tests to predict gonococcal AMR for surveillance and for clinical use, advantages and disadvantages of these tests and of molecular AMR prediction compared with phenotypic AMR testing, and future perspectives for effective use of molecular AMR tests for different purposes. Expert commentary: Several challenges for direct testing of clinical, especially extra-genital, specimens remain. The choice of molecular assay needs to consider the assay target, quality controls, sample types, limitations intrinsic to molecular technologies, and specific to the chosen methodology, and the intended use of the test. Improved molecular- and particularly genome-sequencing-based methods will supplement AMR testing for surveillance purposes, and translate into point-of-care tests that will lead to personalized treatments, while sparing the last available empiric treatment option (ceftriaxone). However, genetic AMR prediction will never completely replace phenotypic AMR testing, which detects also AMR due to unknown AMR determinants

In Vitro Activity of Three Commercial Bacteriophage Cocktails against Multidrug-Resistant Escherichia coli and Proteus spp. Strains of Human and Non-Human Origin

Background: Bacteriophages could represent a therapeutic alternative to treat infections caused by multidrug-resistant (MDR) pathogens. However, studies analyzing their activity against MDR Enterobacteriaceae are limited. Methods: The in vitro lytic activity of three commercial bacteriophage cocktails (PYO, INTESTI, Septaphage) was evaluated against 70 Escherichia coli and 31 Proteus spp. of human and non-human origin. Isolates were characterized by phenotypic and genotypic methods and included 82 MDR strains: 44 ESBL (of which 15 CTX-M-15-like, including ST131/ST648 E. coli), 27 pAmpC (of which 23 CMY-2-like, including ST131 E. coli), 3 ESBL plus pAmpC, and 8 carbapenemase producers. Phage susceptibility was determined using the spot test. Results: E. coli susceptibility to PYO, INTESTI, and Septaphage was 61%, 67%, and 9%, whereas that of Proteus spp. was 29%, 39%, and 19%, respectively. For the subgroup of ESBL-producing E. coli/Proteus spp., the following susceptible rates were recorded: PYO, 57%; INTESTI, 59%; and Septaphage, 11%. With regard to the pAmpC producers, 59%, 70% and 11% resulted susceptible to PYO, INTESTI, and Septaphage, respectively. Five out of 8 carbapenemase producers and 3 out of 4 colistin-resistant E. coli were susceptible to PYO and INTESTI. Conclusions: This is the first study analyzing the activity of the above three cocktails against well-characterized MDR E. coli and Proteus spp. The overall narrow-spectrum of activity observed could be related to the absence of specific bacteriophages targeting these contemporary MDR strains that are spreading in different settings. Therefore, bacteriophages targeting emerging MDR pathogens need to be isolated and integrated in such bio-preparations

In Vitro Activity of 3 Commercial Bacteriophage Cocktails Against Salmonella and Shigella spp. Isolates of Human Origin

Background: Salmonella and Shigella spp. are 2 of the most frequent and deadly enteric bacterial pathogens recorded worldwide. In developing countries Salmonella infections are responsible for many deaths annually and these mortality rates are prone to increase due to the emergence of resistance to antibiotics. In this overall scenario new alternative therapeutic approaches are needed. Methods: For the first time, we investigated the activity of 3 commercial bacteriophage cocktails (INTESTI, Septaphage, PYO) against a collection of contemporary Salmonella spp. (n = 30) and Shigella spp. (n = 20) strains isolated in Switzerland. Phage susceptibility was determined by implementing the spot test. Results: The overall susceptibility of Salmonella spp. to INTESTI and Septaphage was 87% and 77%, respectively. With regard to Shigella spp., the overall susceptibility to INTESTI and Septaphage was 95% and 55%, respectively. PYOwas observed to be active against only 10% of Salmonella spp. but against 95% of Shigella spp. Conclusions: Our results seem promising, especially for the INTESTI biopreparation against Salmonella enterica infections. Nevertheless, such speculation should be supported by further in vivo studies to confirm efficacy and safety of the cocktails. We also emphasize the importance of large in vitro screening analyses aimed to assess the activity of such biopreparations against contemporary multidrug-resistant strains that are emerging worldwide. Keywords: commercial; bacteriophages; Salmonella; Shigella cocktail

Functional Dissection of the PE Domain Responsible for Translocation of PE_PGRS33 across the Mycobacterial Cell Wall

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PERv1818c), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PERv1818c domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation

Continuing evidence that COVID-19 has influenced syphilis epidemiology in Rome

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A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Caratterizzazione dei fattori sigma micobatterici SigE e SigF Caratterizzazione del dominio PPE della proteina PPE17 di Mycobacterium tuberculosis

Abstract Mycobacterium tuberculosis (MTB) is the causative of tuberculosis, a disease, which causes 2 millions of death every year, with a dramatic incidence especially in developing countries. To find new drug and vaccine strategies against MTB, it is of fundamental importance to study the mechanisms, that allow its survival to environmental stresses, to which it is subjected during the period of infection and latency in the host macrophages. The fine transcriptional regulation of specific genes in response to stress conditions and the peculiar structure of its wall play a key role on this. In the first part of the PhD project two mycobacterial sigma factors, SigE and SigF, which regulate the transcription of specific genes in response to various environmental stresses such as surface stress, oxidative stress, alkaline pH and thermal shock, have been characterized. First the transcriptional regulation, translational and post-translational regulation of the extracytoplasmic function (ECF) transcription factor SigE were studied. Regarding the study of the transcriptional regulation, it was possible to confirm by 5'RACE PCR and RT-PCR experiments the presence of three promoters of sigE, and to determine the contribution of each promoter in the transcription of this gene, depending on the environmental conditions of bacterial growth. The fact, that the transcriptional start codon of one of these promoters is located 63 base pairs downstream of the start codon annotated in MTB genome opened the possibility of the existence of two isoforms of Sige. By translational fusions between specific sequences of sigE with lacZ, deprived of its own translational initiation codon, and subsequent site-specific mutagenesis, it was possible to confirm, based on further beta-galactosidase activity detection, the existence of two alternative start codons, an ATC and a TTG, coding for an isoform of respectively 218 and 215 of amino acids, in addition to the ATG already annotated in MTB genome, which encodes for an isoform of 257 amino acids. Finally, it was possible to confirm, that the gene downstream sigE encodes for the anti-sigma factor of SigE, called RseA, capable of binding both isoforms of SigE. In a second project also the role of the factor SigF M. smegmatis in the biosynthesis of carotenoid pigments, resistance to hydrogen peroxide and in the efficiency of bacterial transformation was studied. By RT-PCR it has been shown, that SigF controls the transcription of genes involved in the biosynthesis of carotenoid pigments, and, assuming that they serve as protection against free radicals, it was verified that the sigF mutant strain is actually more sensitive compared to the wild type strain to treatment with hydrogen peroxide. Finally, we also demonstrated, that the mutant strain has a higher transformation efficiency than the wild type strain, indicating that SigF regulates the transcription of genes possibly involved in the permeability of the cell wall. In the second part of the project, the localization of the protein on the surface PPE17 mycobacteria was characterized. Like other members of the PPE family, the PPE17 has a highly conserved N-terminal domain, which, based on different evidences in literature, is assumed to play an important role in their translocation to the mycobacterial surface. Moreover, it was investigated the possible influence of the presence of PE11 in the translocation process or in the stability of PPE17, as the PE11 coding sequence is in tandem and co-transcribed with that encoding the PPE17, and there is a specific interaction between these two proteins. The data obtained by proteinase K sensitivity assays performed on M. smegmatis strains, expressing the entire PPE17 or only its domain PPE (dPPE17) fused with the HA epitope, confirm, that the entire PPE17 is exposed on the surface, both in the presence and absence of PE11. According to data obtained, the possibility to translocate the MTB model antigen (Mpt64) on the surface of the vaccine strain M. bovis BCG, by fusing them with the dPPE17 was tested. Proteinase K and whole cell ELISA assays performed on cultures of M. bovis BCG expressing this chimeric protein indicate, that it is indeed localized at the mycobacterial surface. Similarly, another two fusions with dPPE17 were constructed to express on the mycobacterial surface the multimeric MTB antigen AG85-ESAT6 of MTB and the Csp C3 antigen of Plasmodium bergii. According to the proteinase K sensitivity assays carried out on strains of M. smegmatis expressing the two chimeric proteins indicate that also in this case both are localized at the surface. The strains of M. bovis BCG expressing these antigens on their surface will be tested in future in the mouse model to measure any increase in protection compared to the wild type strain.Riassunto Mycobacterium tuberculosis (MTB) è l’agente eziologico della tubercolosi, patologia che nel mondo causa ogni anno due milioni di morti, con un’incidenza drammatica specie nei Paesi in via di sviluppo. Per poter trovare nuove strategie farmacologiche e vaccinali contro MTB è di fondamentale importanza lo studio dei meccanismi, che permettono la sua sopravvivenza ai vari stress ambientali, ai quali è sottoposto durante il periodo di infezione e latenza nei macrofagi dell’ospite. La fine regolazione della trascrizione di geni specifici in risposta a condizioni di stress e la peculiare struttura della sua parete giocano in merito un ruolo fondamentale. Nella prima parte del progetto di dottorato sono stati caratterizzati due fattori di trascrizione sigma micobatterici, SigE e SigF, che regolano la trascrizione di geni specifici in risposta a vari tipi di stress ambientali, come lo stress di superficie, lo stress ossidativo, il pH alcalino e lo shock termico. Anzitutto è stata studiata la regolazione trascrizionale, traduzionale e posttraduzionale del fattore di trascrizione con funzione extracitoplasmatica (ECF) SigE. Per quanto riguarda lo studio della regolazione trascrizionale, è stato possibile confermare tramite esperimenti di 5’RACE PCR e RT-PCR la presenza di tre promotori di sigE, e a dosare, a seconda delle condizioni ambientali di crescita batterica, il contributo di ciascun promotore nella trascrizione di questo gene. Dato che l’inizio della trascrizione di uno di questi promotori è sito 63 paia di basi a valle del codone di start annotato nel genoma, si è aperta l’ipotesi dell’esistenza di due isoforme di SigE. Mediante fusioni traduzionali tra specifiche sequenze di sigE con lacZ, private del proprio codone di inizio della traduzione, e successive mutagenesi sito-specifiche, è stato possibile confermare, in base all’attività beta-galattosidasica rilevata, l’esistenza di due codoni di start alternativi, un ATC ed un TTG, che codificano per un’isoforma di rispettivamente 218 e 215 di amminoacidi, oltre all’ATG già annotato nel genoma di MTB, che codifica per un’isoforma di 257 amminoacidi. Infine è stato possibile confermare, che il gene a valle di sigE codifica per il fattore anti-sigma di SigE, denominato RseA, in grado di legare entrambe le isoforme di SigE. In un secondo progetto è stato studiato anche il ruolo del fattore SigF di M. smegmatis nella biosintesi di pigmenti carotenoidi, nella resistenza a perossido d’idrogeno e nell’efficienza di trasformazione batterica. Tramite RT-PCR è stato dimostrato che SigF controlla la trascrizione di geni coinvolti nella biosintesi dei pigmenti carotenoidi, e, partendo dal presupposto che essi fungono da protezione contro i radicali liberi, è stato verificato che il mutante per il gene sigF è effettivamente più sensibile rispetto al ceppo selvatico al trattamento con perossido d’idrogeno. Infine è stato dimostrato anche, che il ceppo mutante possiede una maggiore efficienza di trasformazione rispetto al ceppo selvatico, indicando che SigF regola probabilmente la trascrizione di geni coinvolti nella permeabilità della parete. Nella seconda parte del progetto è stata caratterizzata la localizzazione della proteina PPE17 sulla superficie micobatterica. Come altri membri della famiglia PPE, la PPE17 presenta un dominio N-terminale altamente conservato, il quale, in base a diverse evidenze in letteratura, si suppone svolgere un ruolo importante per la loro traslocazione in superficie. Inoltre, si è voluto verificare un’eventuale influenza della presenza della proteina PE11 nel processo di traslocazione in o nella stabilità della PPE17, in quanto la sequenza codificante la PE11 è in tandem e co-trascritta con quella codificante la PPE17, e vi è un’interazione specifica tra queste due proteine. I dati ottenuti mediante saggi di sensibilità alla proteinasi K su ceppi di M. smegmatis, esprimenti la PPE17 intera o solo il suo dominio PPE (dPPE17) fuse all’epitopo HA, confermano che la PPE17 intera sia esposta in superficie, sia in presenza che in assenza di PE11. In base ai dati ottenuti si è infine tentato di veicolare un antigene modello (Mpt64) di MTB sulla superficie del ceppo vaccinale M. bovis BCG fondendolo con il dPPE17. Saggi di sensibilità alla proteinasi K e ELISA su cellule intere effettuati su culture di M. bovis BCG esprimenti questa proteina chimerica indicano, che essa sia effettivamente localizzata a livello superficiale. Allo stesso modo sono state costruite due ulteriori fusioni con il dPPE17 per esprimere sulla superficie micobatterica l’antigene multimerico Ag85-ESAT6 di MTB e l’antigene Csp C3 di Plasmodium berghii. In base a saggi di sensibilità alla proteinasi K svolti su ceppi di M. smegmatis esprimenti le due fusioni anche in questo caso entrambe localizzano in superficie. I ceppi di M. bovis BCG esprimenti questi antigeni sulla loro superficie saranno testati in futuro nel modello del topo per misurare un eventuale aumento della protezione rispetto al ceppo selvatico