Rhamnose-inducible gene expression in Listeria monocytogenes

Acid production from rhamnose is a characteristic phenotype of Listeria monocytogenes. We report the identification of the rhamnose transport and utilization operon located at lmo2846 to lmo2851, including the rhamnose-dependent promoter P(rha). Expression of reporter genes under control of P(rha) on a single copy integration vector demonstrated its suitability for inducible gene expression in L. monocytogenes. Transcription initiation from P(rha) is dose dependent, and a concentration as low as 100 µM rhamnose was found sufficient for induction. Moreover, P(rha) is subject to glucose catabolite repression, which provides additional options for strict control of expression. Infection of human THP1 macrophages revealed that P(rha) is repressed in intracellular L. monocytogenes, which is explained by the absence of rhamnose in the cytosol and possible interference by catabolite repression. The P(rha) promoter provides a novel and useful tool for triggering gene expression in extracellular L. monocytogenes, whereas intracellular conditions prevent transcription from this promoter

Rhamnose-Inducible Gene Expression in Listeria monocytogenes

Acid production from rhamnose is a characteristic phenotype of Listeria monocytogenes. We report the identification of the rhamnose transport and utilization operon located at lmo2846 to lmo2851, including the rhamnose-dependent promoter Prha. Expression of reporter genes under control of Prha on a single copy integration vector demonstrated its suitability for inducible gene expression in L. monocytogenes. Transcription initiation from Prha is dose dependent, and a concentration as low as 100 µM rhamnose was found sufficient for induction. Moreover, Prha is subject to glucose catabolite repression, which provides additional options for strict control of expression. Infection of human THP1 macrophages revealed that Prha is repressed in intracellular L. monocytogenes, which is explained by the absence of rhamnose in the cytosol and possible interference by catabolite repression. The Prha promoter provides a novel and useful tool for triggering gene expression in extracellular L. monocytogenes, whereas intracellular conditions prevent transcription from this promoter.ISSN:1932-620

Rhamnose dependent growth of <i>L. monocytogenes</i> in batch culture.

<p>Panel A: Growth of <i>L. monocytogenes</i> 10403S in LB broth supplemented with glucose (50 mM, open circle) and rhamnose (50 mM, open squares), respectively. When no carbon source was added (closed circles), <i>L. monocytogenes</i> showed only poor growth. Panel B: Growth of <i>L. monocytogenes</i> LF002 (<i>ermC</i> under control of P<i>_rha</i>) in medium supplemented with 7.5 µg/ml erythromycin. LF002 cells pre-induced with rhamnose (open squares) showed a slight delay in growth response, whereas non-induced cells (closed circles) did not multiply. <i>L. monocytogenes</i> strain LF003 (constitutive <i>ermC</i> expression) was used as positive control (open circles).</p

Intracellular multiplication of <i>prfA</i>-negative and P<i>_rha</i>-dependent trans-complemented <i>L. monocytogenes</i> after infection of human THP1 macrophages.

<p>wt: wild type; Δ<i>prfA</i>: <i>prfA</i> knock out; LF006 +: single copy <i>prfA</i> under control of P<i>_rha</i> (pre-induced overnight with 10 mM rhamnose); LF006 −: non-induced.</p

Rhamnose-inducible expression of GFP.

<p>Panels A and B: Dose-dependent response of P<i>_rha</i>-controlled <i>gfp</i> expression in <i>L. monocytogenes</i> LF001 after 16 h of induction using rhamnose concentrations as indicated. Panel A, top row: fluorescence microscopy; bottom row, phase contrast microscopy. Positive and negative controls, and the effect of catabolite repression by addition of equimolar amounts of glucose and rhamnose are indicated. rha: rhamnose, glc: glucose. Panel B: quantitation of relative fluorescence (RFU) of GFP in rhamnose-induced bacteria. Positive (P_hyp) and negative (no promoter) controls are indicated on the left. Addition of 100 µM rhamnose increased fluorescence significantly (p<0.001). Panel C: quantitative catabolite repression of P<i>_rha</i>-dependent <i>gfp</i> expression in the presence of rhamnose together with a second carbohydrate (indicated on the x-axis), at equimolar concentration (10 mM). Positive (P_hyp) and negative (no promoter) controls are indicated on the right.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

The <i>L. monocytogenes</i> rhamnose utilization operon.

<p>Dark grey: open reading frames <i>lmo2846-lmo2851</i> located on both DNA strands <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043444#pone.0043444-Glaser1" target="_blank">[9]</a> and their functional assignments; white: P<i>_rha</i> promoter, putative -10 and -35 regions are shaded; black: transcription terminators.</p

How frequent is routine use of probiotics in UK neonatal units?

Objective There is a lack of UK guidance regarding routine use of probiotics in preterm infants to prevent necrotising enterocolitis, late-onset sepsis and death. As practices can vary, we aimed to determine the current usage of probiotics within neonatal units in the UK.Design and setting Using NeoTRIPS, a trainee-led neonatal research network, an online survey was disseminated to neonatal units of all service levels within England, Scotland, Northern Ireland and Wales in 2022. Trainees were requested to complete one survey per unit regarding routine probiotic administration.Results 161 of 188 (86%) neonatal units responded to the survey. 70 of 161 (44%) respondents routinely give probiotics to preterm infants. 45 of 70 (64%) use the probiotic product Lactobacillus acidophilus NCFM/Bifidobacterium bifidum Bb-06/B. infantis Bi-26 (Labinic™). 57 of 70 (81%) start probiotics in infants ≤32 weeks’ gestation. 33 of 70 (47%) had microbiology departments that were aware of the use of probiotics and 64 of 70 (91%) had a guideline available. Commencing enteral feeds was a prerequisite to starting probiotics in 62 of 70 (89%) units. The majority would stop probiotics if enteral feeds were withheld (59 of 70; 84%) or if the infant was being treated for necrotising enterocolitis (69 of 70; 99%). 24 of 91 (26%) units that did not use probiotics at the time of the survey were planning to introduce them within the next 12 months.Conclusions More than 40% of all UK neonatal units that responded are now routinely administering probiotics, with variability in the product used. With increased probiotic usage in recent years, there is a need to establish whether this translates to improved clinical outcomes

Comparison of diagnoses of early-onset sepsis associated with use of Sepsis Risk Calculator versus NICE CG149: a prospective, population-wide cohort study in London, UK, 2020–2021

Objective We sought to compare the incidence of early-onset sepsis (EOS) in infants ≥34 weeks’ gestation identified &gt;24 hours after birth, in hospitals using the Kaiser Permanente Sepsis Risk Calculator (SRC) with hospitals using the National Institute for Health and Care Excellence (NICE) guidance.Design and setting Prospective observational population-wide cohort study involving all 26 hospitals with neonatal units colocated with maternity services across London (10 using SRC, 16 using NICE).Participants All live births ≥34 weeks’ gestation between September 2020 and August 2021.Outcome measures EOS was defined as isolation of a bacterial pathogen in the blood or cerebrospinal fluid (CSF) culture from birth to 7 days of age. We evaluated the incidence of EOS identified by culture obtained &gt;24 hours to 7 days after birth. We also evaluated the rate empiric antibiotics were commenced &gt;24 hours to 7 days after birth, for a duration of ≥5 days, with negative blood or CSF cultures.Results Of 99 683 live births, 42 952 (43%) were born in SRC hospitals and 56 731 (57%) in NICE hospitals. The overall incidence of EOS (&lt;72 hours) was 0.64/1000 live births. The incidence of EOS identified &gt;24 hours was 2.3/100 000 (n=1) for SRC vs 7.1/100 000 (n=4) for NICE (OR 0.5, 95% CI (0.1 to 2.7)). This corresponded to (1/20) 5% (SRC) vs (4/45) 8.9% (NICE) of EOS cases (χ=0.3, p=0.59). Empiric antibiotics were commenced &gt;24 hours to 7 days after birth in 4.4/1000 (n=187) for SRC vs 2.9/1000 (n=158) for NICE (OR 1.5, 95% CI (1.2 to 1.9)). 3111 (7%) infants received antibiotics in the first 24 hours in SRC hospitals vs 8428 (15%) in NICE hospitals.Conclusion There was no significant difference in the incidence of EOS identified &gt;24 hours after birth between SRC and NICE hospitals. SRC use was associated with 50% fewer infants receiving antibiotics in the first 24 hours of life