A Pan-European Review of Good Practices in Early Intervention Safeguarding Practice with Children, Young People and Families: Evidence Gathering to Inform a Multi-disciplinary Training Programme (the ERICA Project) in Preventing Child Abuse and Neglect in Seven European Countries.

Child maltreatment has detrimental social and health effects for individuals, families and communities. The ERICA project is a pan-European training programme that equips non-specialist threshold practitioners with knowledge and skills to prevent and detect child maltreatment. This paper describes and presents the findings of a rapid review of good practice examples across seven participating countries including local services, programmes and risk assessment tools used in the detection and prevention of child maltreatment in the family. Learning was applied to the development of the generic training project. A template for mapping the good practice examples was collaboratively developed by the seven participating partner countries. A descriptive data analysis was undertaken organised by an a priori analysis framework. Examples were organised into three areas: programmes tackling child abuse and neglect, local practices in assessment and referral, risk assessment tools. Key findings were identified using a thematic approach. Seventy-two good practice examples were identified and categorised according to area, subcategory and number. A typology was developed as follows: legislative frameworks, child health promotion programmes, national guidance on child maltreatment, local practice guidance, risk assessment tools, local support services, early intervention programmes, telephone or internet-based support services, COVID-19 related good practices. Improved integration of guidance into practice and professional training in child development were highlighted as overarching needs. The impact of COVID-19 on safeguarding issues was apparent. The ERICA training programme formally responded to the learning identified in this international good practice review.

Mutational analyses of open reading frames within the vraSR operon and their roles in the cell wall stress response of Staphylococcus aureus.

The exposure of Staphylococcus aureus to a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. The vraSR genes form part of the four-cistron autoregulatory operon orf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed that orf1 played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF in S. aureus appears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.

Transcriptional profiling of XdrA, a new regulator of spa transcription in Staphylococcus aureus.

Transcription of spa, encoding the virulence factor protein A in Staphylococcus aureus, is tightly controlled by a complex regulatory network, ensuring its temporal expression over growth and at appropriate stages of the infection process. Transcriptomic profiling of XdrA, a DNA-binding protein that is conserved in all S. aureus genomes and shares similarity with the XRE family of helix-turn-helix, antitoxin-like proteins, revealed it to be a previously unidentified activator of spa transcription. To assess how XdrA fits into the complex web of spa regulation, a series of regulatory mutants were constructed; consisting of single, double, triple, and quadruple mutants lacking XdrA and/or the three key regulators previously shown to influence spa transcription directly (SarS, SarA, and RNAIII). A series of lacZ reporter gene fusions containing nested deletions of the spa promoter identified regions influenced by XdrA and the other three regulators. XdrA had almost as strong an activating effect on spa as SarS and acted on the same spa operator regions as SarS, or closely overlapping regions. All data from microarrays, Northern and Western blot analyses, and reporter gene fusion experiments indicated that XdrA is a major activator of spa expression that appears to act directly on the spa promoter and not through previously characterized regulators.

Variability of SCCmec in the Zurich area.

A periodic survey of methicillin-resistant Staphylococcus aureus (MRSA) in Zurich in 2004 and 2006 revealed a consistently low prevalence of MRSA. SCCmec and ccr typing showed fluctuations in the proportions of SCCmec types and in the carriage of mobile virulence determinants. Together with the presence of variant SCCmecs these findings suggest a high clonal diversity and level of SCCmec recombination. The prevalence of a local "drug clone", associated with low-level methicillin resistance and rapid growth, significantly decreased. This clone had spread among intraveneous drug users, steadily increasing from 1994 to 2001 and was dominant in 2001. Apparently, changes in the management of the Zurich drug scene have restricted the spread of this clone.

Strain dependence of the cell wall-damage induced stimulon in Staphylococcus aureus.

The vancomycin stress induced transcriptome of the methicillin susceptible Staphylococcus aureus (MSSA) strain Newman was determined by microarray analysis. Subsets of the induced ORFs corresponded to those previously reported to be induced by vancomycin in the methicillin resistant S. aureus (MRSA) strains N315 and JH1, and/or by other cell wall active antibiotics in RN450; while other ORFs appeared to be induced strain specifically in Newman. Northern analyses showed that the induction pathway for several of the ORFs appeared to be altered in a number of clinical NARSA isolates. Induction was found to be dependent on inhibitory concentrations of antibiotics.

Comparative genomics and DNA array-based genotyping of pandemic Staphylococcus aureus strains encoding Panton-Valentine leukocidin.

Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.

Expression of resistance to methicillin.

Methicillin-resistant staphylococci have an additional low-affinity penicillin-binding protein, PBP2a (PBP2'), encoded by the mecA gene. The typical heterogeneity seen in the expression of resistance to methicillin and in levels of resistance depends on the concerted action of chromosomally encoded genes, including fem and aux, that are also present in the genome of susceptible staphylococci.

Inactivation of a novel three-cistronic operon tcaR-tcaA-tcaB increases teicoplanin resistance in Staphylococcus aureus.

A novel teicoplanin-associated operon termed tcaR-tcaA-tcaB was identified by Tn917-mediated insertional mutagenesis. Resistance to teicoplanin rose 4-fold by insertional inactivation of tcaA or by deletion of the entire operon. tcaA encodes a hypothetical transmembrane protein with a metal-binding motif, possibly a sensor-transducer. tcaB codes for a membrane-associated protein, which has sequence homologies to a bicyclomycin resistance protein. The two genes are preceded by tcaR encoding a putative regulator with sequence homologies to the transcriptional regulator MarR. The fact that tcaA inactivation as well as deletion of tcaRAB produced the same increase in teicoplanin resistance confirmed the association of tcaRAB with teicoplanin susceptibility. Cotransductional crosses showed that the level of teicoplanin resistance produced by these insertions was strain-dependent and that in the methicillin-resistant strain COL, it was paired with a remarkable decrease in methicillin resistance. This allowed to postulate that tcaRAB may be involved in some way in cell wall biosynthesis, and that teicoplanin may interact with TcaA and/or TcaB either directly or indirectly.

DNA restriction--modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies.

We have purified the type III restriction enzymes EcoP1 and EcoP15 to homogeneity from bacteria that contain the structural genes for the enzymes cloned on small, multicopy plasmids and which overproduce the enzymes. Both of the enzymes contain two different subunits. The molecular weights of the subunits are the same for both enzymes and antibodies prepared against one enzyme cross-react with both subunits of the other. Bacteria containing a plasmid derivative in which a large part of one of the structural genes has been deleted have a restriction- modification+ phenotype and contain only the smaller of the two subunits. This subunit therefore must be the one that both recognizes the specific DNA sequence and methylates it in the modification reaction (the restriction enzyme itself also acts as a modification methylase). We have purified the P1 and P15 modification subunits from these deletion derivatives and have shown that in vitro they have the expected properties: they are sequence-specific modification methylases. In addition, we have demonstrated that strains carrying the full restriction/modification system also contain a pool of free modification subunits that might be responsible for in vivo modification.

DNA restriction--modification genes of phage P1 and plasmid p15B. Structure and in vitro transcription.

The EcoP1 and EcoP15 DNA restriction-modification systems are coded by the related P1 prophage and p15B plasmid. We have examined the organization of the genes for these systems using P1 itself, "P1-P15" hybrid phages expressing the EcoP15 restriction specificity of p15B and cloned restriction fragments derived from these phage DNAs. The results of transposon mutagenesis, restriction cleavage analysis. DNA heteroduplex analysis and in vitro transcription mapping allow the following conclusions to be drawn concerning the structural genes. (1) All of the genetic information necessary to specify either system is contained within a contiguous DNA segment of 5 x 10(3) bases which encodes two genes. One of them, necessary for both restriction and modification, we call mod and the other, required only for restriction (together with mod), we call res. (2) The res gene is about 2.8 x 10(3) bases long and at the heteroduplex level is largely identical for P1 and P15: it shows a small region of partial nonhomology and some restriction cleavage site differences. The mod gene is about 2.2 x 10(3) bases long and contains a 1.2 x 10(3) base long region of non-homology between P1 and P15 toward the N-terminus of the gene. The rest of the gene at this level of analysis is identical for the two systems. (3) Each of the genes is transcribed in vitro from its own promoter. It is possible that the res gene is also transcribed by readthrough from the mod promoter.

Sequence of the putative alanine racemase operon in Staphylococcus aureus: insertional interruption of this operon reduces D-alanine substitution of lipoteichoic acid and autolysis.

A gene cluster comprising the alanine racemase gene alr was identified 5' to the sigB operon in Staphylococcus aureus. It is flanked upstream by four ORFs of which one shows similarity to the dpj gene of Escherichia coli, and downstream by two ORFs of which the last shows similarity to the E. coli pemK gene. Preliminary data suggest that the seven ORFs orf1-orf2-orf3-dpj-alr-orf6-pemK may form an operon. Disruption of the proposed operon by insertional mutagenesis leads to a drastic loss in the d-alanine (d-Ala) substitution of lipoteichoic acid and to delayed autolysis, without affecting the d-Ala substitution of the wall teichoic acid.

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris.

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.

Effects of penicillin-binding protein 4 overproduction in Staphylococcus aureus.

The pbp4 gene of a Staphylococcus aureus strain selected stepwise in vitro for growth on increasing concentrations of penicillin and of its susceptible parent strain showed overall identity except in the promoter region. In the mutant a deletion upstream of the pbp4 structural gene removed 90 nucleotides (nt) that were framed by a 12 nt inverted repeat. This deletion occurred in step 4 of the in vitro selection procedure and was paralleled by a significant increase in the penicillin-binding protein 4 (PBP4) production. The in vitro step selected mutant showed a remarkable increase in the cross-linking of the peptidoglycan compared to its parent. This was linked to morphological changes in the appearance of the cells, which were surrounded by a very thick and fuzzy cell wall.

Glutamine synthetase and heteroresistance in methicillin-resistant Staphylococcus aureus.

Inactivation of femC in methicillin-resistant Staphylococcus aureus (MRSA) results in lowered methicillin resistance and a reduction in the amidation of the iso-D-glutamate of the peptidoglycan stem peptide. The femC phenotype is due to insertional inactivation of the glutamine synthetase repressor gene glnR by Tn551, which has a polar effect on glutamine synthetase (glnA) transcription. The complete glutamine synthetase operon (glnRA) of S. aureus was cloned and sequenced, and its transcriptional start was determined. The deduced amino acid sequence of the staphylococcal glutamine synthetase showed 76% identity and 87% similarity to the Bacillus subtilis glutamine synthetase. The staphylococcal glnRA operon was shown to complement an Escherichia coli glutamine synthetase-negative mutant and to restore methicillin resistance in femC mutants. femC mutants revert to resistance in the presence of high concentrations of methicillin. These revertants, which still carried the femC lesion, were shown to retain the lowered amidation of the iso-D-glutamate peptidoglycan stem peptide. A new chromosomal locus hmrC was postulated to have mutated to allow expression of high methicillin resistance in these femC revertants. Although the highly resistant hmrC revertant resembled phenotypically the highly methicillin-resistant subclones occurring in heterogeneously resistant MRSA, we could show by transduction that the locus hmrC was distinct from chr*, a chromosomal site postulated to confer high methicillin resistance in heterogeneous MRSA. This suggests that S. aureus can adopt multiple ways to achieve high methicillin resistance.

Lif, the lysostaphin immunity factor, complements FemB in staphylococcal peptidoglycan interpeptide bridge formation.

The formation of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide chain needs FemA and FemB for the incorporation of glycines Gly2-Gly3, and Gly4-Gly5, respectively. The lysostaphin immunity factor Lif was able to complement FemB, as could be shown by serine incorporation and by an increase in lysostaphin resistance in the wild-type as well as in a femB mutant. However, Lif could not substitute for FemA in femA or in femAB-null mutants. Methicillin resistance, which is dependent on functional FemA and FemB, was not complemented by Lif, suggesting that serine-substituted side chains are a lesser substrate for penicillin-binding protein PBP2' in methicillin resistance.

Impact of sar and agr on methicillin resistance in Staphylococcus aureus.

The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.

Identification of three additional femAB-like open reading frames in Staphylococcus aureus.

Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptide.

Involvement of multiple genetic loci in Staphylococcus aureus teicoplanin resistance.

Teicoplanin resistance was transformed from a teicoplanin-resistant Staphylococcus aureus into the susceptible strain BB255 to give strain BB938. The cell wall composition, amidation of the iD-glutamate, and peptide crosslinking were identical in BB938 as in BB255 except for a 60% increased length of the glycan chain. Transductional crosses revealed that at least two distinct loci contributed in a cumulative fashion to teicoplanin resistance. One of these loci correlated with a mutation inactivating the anti-sigma factor RsbW. This mutation must have occurred during transformation and selection for teicoplanin resistance in BB938. Genetic manipulations involving the sigB operon showed that transcription factor SigB contributed to decreased teicoplanin susceptibility.

Factors affecting methicillin resistance in Staphylococcus aureus.

Methicillin resistance in staphylococci is primarily due to the methicillin resistance determinant which produces an additional low affinity penicillin-binding protein PBP2'. Expression of PBP2' is controlled by its own regulatory element, and shares also the same induction system as beta-lactamases. However, methicillin resistance levels do not correlate with the amount of PBP2' produced, but are determined by the strain specific genetic background. These factors responsible for high methicillin resistance have not yet been characterised.

Identification and mapping of new chromosomal sites affecting response to beta-lactams in Staphylococcus aureus.

Seven new sites leading to increased beta-lactam susceptibility were identified and mapped in Staphylococcus aureus by Tn551 or Tn917 mediated insertional inactivation. Their effect was more pronounced in methicillin resistant than in susceptible strains. Except for inserts each in SmaI-K and SmaI-B, all were located on SmaI-A, which covers 25% of the staphylococcal chromosome. The physical position of the femAB operon involved in the expression of methicillin resistance was mapped on SmaI-A. Close to this site we identified a further site with almost as strong an effect on methicillin resistance as femAB. A second cluster of sites affecting methicillin resistance was identified at approximately 30 kb from the femAB operon, showing that multiple unlinked factors may affect beta-lactam resistance. Interestingly, insert Omega2016 located in SmaI-B produced small colonies and was found to inactivate a gene of the electron transport chain, reminiscent of small colony variants observed in recurrent infections.