Noise in timing and precision of gene activities in a genetic cascade

Biological developmental pathways require proper timing of gene expression. We investigated timing variations of defined steps along the lytic cascade of bacteriophage λ. Gene expression was followed in individual lysogenic cells, after induction with a pulse of UV irradiation. At low UV doses, some cells undergo partial induction and eventually divide, whereas others follow the lytic pathway. The timing of events in cells committed to lysis is independent of the level of activation of the SOS response, suggesting that the lambda network proceeds autonomously after induction. An increased loss of temporal coherence of specific events from prophage induction to lysis is observed, even though the coefficient of variation of timing fluctuations decreases. The observed temporal variations are not due to cell factors uniformly dilating the timing of execution of the cascade. This behavior is reproduced by a simple model composed of independent stages, which for a given mean duration predicts higher temporal precision, when a cascade consists of a large number of steps. Evidence for the independence of regulatory modules in the network is presented

Genomic Epidemiology of Campylobacter jejuni Transmission in Israel

Objectives:Campylobacter jejuni is responsible for 80% of Campylobacter infections in Israel, a country with a high incidence reaching 91/100,000 population. We studied the phylogeny, diversity and prevalence of virulence factors using whole genome sequencing (WGS) of a national sample of C. jejuni clinical, food, and animal isolates collected over a 10-year period (2003–2012).Methods:C. jejuni isolates (n = 263) were subject to WGS using Illumina sequencing (PE 250bpx2). Raw reads and de novo assemblies were analyzed with the BioNumerics whole genome MLST (wgMLST) pipeline. Reads were screened for 71 virulence genes by the SRST2 script. Allelic profiles were analyzed to create minimum spanning trees and allelic core distances were investigated to determine a reliable cutoff for strain determination.Results: wgMLST analysis of 263 C. jejuni isolates indicated significant diversity among the prevalent clonal complexes (CCs) with CC-21 and CC-353 being the most diverse, and CC-574 the most clonal. Within CC-21, sequence type (ST)-1359 created a separate clade. Human, poultry and bovine isolates clustered together across the different STs. Forty four percent of studied isolates were assigned to 29 genetic clusters. Temporal and geographical relatedness were found among the minority of clusters, while most phylogenetically associated cases appeared diffuse and unassociated epidemiologically. The majority of virulence factors were highly prevalent across the dataset and not associated with genotype, source of isolation or invasiveness. Conversely, all 13 genes associated with type VI secretion system (T6SS) were lineage-related and identified in only 18% of the isolates. T6SS was detected in 95.2% of ST-1359, a common type in Israel.Conclusions: wgMLST supported the assessment that poultry and cattle are likely food sources of infection in Israel. Substantial genetic clustering among C. jejuni isolates suggested multiple point source and diffuse outbreaks that were previously unreported in Israel. The high prevalence of T6SS among ST-1359 isolates is unique to Israel, and requires further investigation. This study exemplifies the importance of studying foodborne pathogens using advanced genomic approaches across the entire spectrum of One Health

Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision

The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic α helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell

Trends in invasive bacterial diseases during the first 2 years of the COVID-19 pandemic: analyses of prospective surveillance data from 30 countries and territories in the IRIS Consortium.

BACKGROUND The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. METHODS For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. FINDINGS Overall, 116 841 cases were analysed: 76 481 in 2018-19, before the pandemic, and 40 360 in 2020-21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40-0·55), H influenzae (0·51; 0·40-0·66) and N meningitidis (0·26; 0·21-0·31), while no significant changes were observed for S agalactiae (1·02; 0·75-1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145-55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. INTERPRETATION COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. FUNDING Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization

Epidemiological and Clinical Characteristics of Non-Typhoidal <i>Salmonella</i> Bloodstream Infections in Central Israel: A Case-Control Study

Non-typhoidal Salmonella (NTS) infection continues to be a significant cause of morbidity. In addition to gastroenteritis (GE), NTS may cause bloodstream infections (BSI). Our goals were to characterize the demographics, clinical characteristics and outcome of NTS-BSI in central Israel. The study was a retrospective, case-control study conducted at the Tel Aviv Sourasky Medical Center between 2001–2018. Cases with NTS-BSI were matched by age and compared with two control groups, hospitalized patients with NTS-GE and patients with E. coli BSI. The NTS-BSI group included 34 patients who were compared with 69 and 68 patients in the NTS-GE and E. coli BSI groups, respectively. In the NTS-BSI group, the median age was 59 years, with 20% of patients below 20 years of age. Diarrhea was less common in NTS-BSI patients compared with NTS-GE: 53% vs. 80% (p p = 0.03, respectively. They also had a slightly higher Charlson Comorbidity Index score, and history of past malignancy and steroid use, but these differences were not statistically significant. Antimicrobial treatment was documented in 30/34 of the NTS-BSI patients vs. 55/69 of the NTS-GE patients (p p p E. coli BSI group. In conclusion, our study found relatively low rates of pediatric cases compared with previous studies in Israel. NTS-BSI patients had slightly higher rates of comorbidities compared with NTS-GE patients, and a similar prognosis to E. coli BSI patients

Ler Is a Negative Autoregulator of the LEE1 Operon in Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes

Hospital clones of methicillin-resistant Staphylococcus aureus are carried by medical students even before healthcare exposure

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) strains are prevalent in healthcare and the community. Few studies have examined MRSA carriage among medical students. The aim of this study is to examine Staphylococcus aureus (SA) carriage, and particular MRSA, over time in cohort medical students Methods Prospective collection of nasal swabs from medical students in Israel and assessment of SA carriage. Three samples were taken per student in preclinical and clinical parts of studies. Antibiotic susceptibilities were recorded and MRSA typing was performed by staphylococcal cassette chromosome mec (SCCmec) types, Panton Valentine Leukocidin (PVL) encoding genes, and spa types. Clonality was assessed by pulsed-field gel electrophoresis. Results Among 58 students, SA carriage rates increased from 33% to 38% to 41% at baseline (preclinical studies), 13 and 19 months (clinical studies), respectively (p = 0.07). Methicillin-susceptible SA (MSSA) carriage increased in the clinical studies period (22 to 41%, p = 0.01). Overall, seven students (12%) carried 13 MRSA isolates. MRSA isolates were PVL negative and were characterized as SCCmecII-t002, SCCmecIV-t032, or t12435 with untypable SCCmec. MRSA carriage during the pre-clinical studies was evident in 4/7 students. Two students carried different MRSA clones at various times and persistent MRSA carriage was noted in one student. Simultaneous carriage of MRSA and MSSA was not detected. Conclusions MSSA carriage increased during the clinical part of studies in Israeli medical students. Compared with previous reports, higher rates of MRSA carriage were evident. MRSA strains were genotypically similar to Israeli healthcare-associated clones; however, carriage occurred largely before healthcare exposure, implying community-acquisition of hospital strains

Circular dichroism of CIII.

<p>(A) 200 pmol of purified His₆-CIII (right lane) were loaded on 4–12% NuPAGE (invitrogen) and visualized by Coomassie staining. On the left lane protein size marker was loaded (sizes 49, 38, 28, 14, 6 and 3 kDa). (B) The Circular dichroism spectrum (wavelengths 195–255 nm) of the purified His₆-CIII at 3.5 µM (light gray), 27 µM (dark gray) and 40 µM (black).</p

CIII levels effect on CII and Q activity <i>in vivo.</i>

<p>CII activity is reported by pE-<i>gfp</i> fusions (blue diamonds), whereas Q activity is reported by pR‘-tR‘-<i>gfp</i> fusions (red circles). Promoter activity is given as a function of time of infected cultures carrying the pCTCIII plasmid in the presence or absence of 0.1mM IPTG (B, D, F or A, C, E respectively) with λ<i>c+</i> (A, B), λ<i>cIII⁻</i> (C, D), and λ<i>cII⁻</i> (E, F). All measurements were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000363#s4" target="_blank">materials and methods</a> at a MOI of 6.</p

Conservation of CIII in phage and bacteria.

<p>The multi sequences alignment was done using the ClustalX (version 1.81) program. Conserved or partially conserved residues are colored according to their biochemical properties. The essential region for CIII activity is underlined and the positions predicted coiled coil (by the MARCOIL) is marked above the sequence. (A) Alignment of CIII proteins from different phages and prophages (represented by the phage name or the bacterial strain carrying the prophage, respectively). CIII proteins were collected from the BLAST program by using the lambda CIII sequence as a query for the non-redundant database. A separate search using the HK022 CIII sequence was done. The HK022 CIII sequence is identical to that of HK097A and H-19B. A complete list of all the phages and prophages in which CIII homologs were recognized, is presented in the supplementary material. (B) Alignment of CIII and SpoVM proteins was carried out with the lambda CIII protein aligned with three SpoVM proteins from different Bacillus strains. (C) Alignment of CIII and YjhS hypothetical proteins that carry the DUF1737 and DUF303, was carried out. The three YjhS-like proteins were derived from different Shigella toxin carrying phages. The HP stands for hypothetical protein without a specific name.</p