Streptococcus pyogenes is a leading cause of infection-related mortality in humans globally. The characteristic cell wall-anchored group A carbohydrate (GAC) is expressed by all S. pyogenes strains and consists of a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains, of which 25% are decorated with glycerol phosphate (GroP). The genes in the gacA-L cluster are critical for GAC biosynthesis, with gacH-L being responsible for the characteristic GlcNAc-GroP decoration, which confers the agglutination in rapid test diagnostic assays and contributes to S. pyogenes pathogenicity. Historical research papers described S. pyogenes isolates, so-called A-variant strains, that lost the characteristic GlcNAc side chain following serial animal passage. Genomic analysis of a single viable historic parent/A-variant strain pair revealed a premature inactivating stop codon in gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analysed the genetic variation of the 12 gacA-L genes in a collection of 2021 S. pyogenes genome sequences. Although all gac genes (gacA-L) displayed genetic variation, we only identified 26 isolates (1.3%) with a premature stop codon in one of the gac genes. Twelve out of 26 (46%) isolates contained a premature stop codon in gacH, which encodes the enzyme responsible for the GroP modification. To study the functional consequences of the different premature stop codons for GacH function, we plasmid-expressed three gacH variants in a S. pyogenes gacH-deficient strain. Cell wall analysis confirmed GacH loss of function for the studied gacH variants through the significant reduction of GAC GroP, complete resistance to killing by the human bactericidal enzyme group IIA-secreted phospholipase and susceptibility to zinc toxicity. Overall, our data provide a comprehensive overview of the genetic variation of the gacA-L cluster in a global population of S. pyogenes strains and the functional consequences of rare inactivating mutations in gacH for host interaction.

ABSTRACTPhase-variable type I restriction modification (RM) systems are epigenetic regulatory systems that have been identified in numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage of Streptococcus suis acquired a phase-variable type I RM system, named SsuCC20p. The SsuCC20p locus was identified in the genome of multiple streptococcal species, indicating that it is not restricted to S. suis and can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase variability relies on a recombinase present within the locus. SsuCC20p is the single RM system responsible for the genome methylation profiles that were detected in the representative zoonotic S. suis isolate 861160 in vitro. In addition, we show that, contrary to previous observations, hsdS genes located downstream of hsdM and the recombinase gene contribute to the SsuCC20p genome methylation profile. SsuCC20p locked mutants expressing a single hsdS each show a unique genome methylation profile and, when grown in human serum, have distinct transcriptomes. In a zebrafish larvae infection model, we observed significant differences in virulence between the locked mutants and a corresponding shift in hsdS allele distribution in the wild type. These data indicate that the streptococcal phase-variable type I RM system SsuCC20p can impact bacterial fitness via epigenetic regulation of gene expression, which impacts the virulence of S. suis in the zebrafish larvae infection model.IMPORTANCEPhase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

Phase-variable Type I Restriction Modification (RM) systems are epigenetic regulatory systems that have been identified in numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage of Streptococcus suis acquired a phase-variable Type I RM system named SsuCC20p. The SsuCC20p locus was present in the genome of disease-associated isolates from multiple streptococcal species. This indicates that it is not restricted to S. suis and can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase-variability relies on a recombinase present within the locus. In vitro , only SsuCC20p is responsible for the genome methylation profiles that were detected in the representative zoonotic S. suis isolate 861160. In addition, we show that, contrary to previous observations, hsdS genes located downstream of the hsdM gene and the recombinase gene, can contribute to the SsuCC20p genome methylation profile. SsuCC20p locked mutants expressing a single hsdS each showed unique genome methylation profiles. The differential genome methylation of the distinct locked mutants caused phase dependent differences in global gene expression in a growth condition dependent manner. We observed significant differences in virulence between hsdS locked mutants in a zebrafish larvae infection model. These data indicate that the streptococcal phase-variable Type I RM system SsuCC20p can impact bacterial virulence via epigenetic regulation of gene expression and potentially contributes to the zoonotic potential of S. suis . Importance Phase-variation contributes to the virulence of bacterial pathogens as it allows a single strain to produce phenotypic diverse subpopulations. Phase-variable Restriction Modification (RM) systems are systems that allow for such phase-variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis . We show that the phase-variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and showed that the differential genome methylation within the phases causes differences in gene expression levels and virulence. Altogether, we show that the acquisition of a phase-variable RM system impacts virulence and can potentially contribute to the zoonotic potential of S. suis . Bacterial pathogens can increase their virulence through acquisition of mobile elements containing epigenetic regulatory systems such as RM systems.

Neisseria meningitidis (meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small noncoding RNAs (sRNAs) are used to fine-tune expression of genes, allowing adaptation to their physiological differences. We have previously characterized sRNAs (Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here, we extend the NmsR regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a ΔnmsRs strain. Levels of transcription of target genes and NmsRs were assessed by reverse transcriptase quantitative PCR (RT-qPCR). We also used a novel gene reporter system in which the 5' untranslated region (5' UTR) of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus. Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1 by direct interaction with the 5' UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. IMPORTANCE Neisseria meningitidis colonizes the human nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that coinhabit the human nasopharynx can be utilized by meningococci for replication in this environment. Here, we showed that sibling small RNAs, designated NmsRs, riboregulate propionic acid utilization by meningococci and, thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs are downregulated. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

Neisseria meningitidis or the meningococcus, can cause devasting diseases such as sepsis and meningitis. Its polysaccharide capsule, on which serogrouping is based, is the most important virulence factor. Non-encapsulated meningococci only rarely cause disease, due to their sensitivity to the host complement system. How the capsular polysaccharide structure of N. meningitidis relates to virulence is largely unknown. Meningococcal virulence can be modeled in zebrafish embryos as the innate immune system of the zebrafish embryo resembles that of mammals and is fully functional two days post-fertilization. In contrast, the adaptive immune system does not develop before 4 weeks post-fertilization. We generated isogenic meningococcal serogroup variants to study how the chemical composition of the polysaccharide capsule affects N. meningitidis virulence in the zebrafish embryo model. H44/76 serogroup B killed zebrafish embryos in a dose-dependent manner, whereas the non-encapsulated variant was completely avirulent. Neutrophil depletion was observed after infection with encapsulated H44/76, but not with its non-encapsulated variant HB-1. The survival of embryos infected with isogenic capsule variants of H44/76 was capsule specific. The amount of neutrophil depletion differed accordingly. Both embryo killing capacity and neutrophil depletion after infection correlated with the number of carbons used per repeat unit of the capsule polysaccharide during its biosynthesis (indicative of metabolic cost).Conclusion:Meningococcal virulence in the zebrafish embryo largely depends on the presence of the polysaccharide capsule but the extent of the contribution is determined by its structure. The observed differences between the meningococcal isogenic capsule variants in zebrafish embryo virulence may depend on differences in metabolic cost.

Staphylococcus aureus is a leading cause of skin and soft tissue infections and systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, phage-mediated horizontal gene transfer, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with β-linked GlcNAc at the C-4 (β1,4-GlcNAc) and the C-3 position (β1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with α-linked GlcNAc at the C-4 position (α1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally present within the S. aureus population, whereas a smaller proportion co-contain either tarM or tarP. To gain more insight into the presence and genetic variation of tarS, tarM and tarP in the S. aureus population, we analysed a collection of 25 652 S. aureus genomes within the PubMLST database. Over 99 % of isolates contained tarS. Co-presence of tarS/tarM or tarS/tarP occurred in 37 and 7 % of isolates, respectively, and was associated with specific S. aureus clonal complexes. We also identified 26 isolates (0.1 %) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

ABSTRACT Neisseria meningitidis (the meningococcus) is primarily a commensal of the human oropharynx that sporadically causes septicemia and meningitis. Meningococci adapt to diverse local host conditions differing in nutrient supply, like the nasopharynx, blood, and cerebrospinal fluid, by changing metabolism and protein repertoire. However, regulatory transcription factors and two-component systems in meningococci involved in adaptation to local nutrient variations are limited. We identified novel sibling small regulatory RNAs (Neisseriametabolic switch regulators [NmsRs]) regulating switches between cataplerotic and anaplerotic metabolism in this pathogen. Overexpression of NmsRs was tolerated in blood but not in cerebrospinal fluid. Expression of six tricarboxylic acid cycle enzymes was downregulated by direct action of NmsRs. Expression of the NmsRs themselves was under the control of the stringent response through the action of RelA. Small sibling regulatory RNAs of meningococci, controlling general metabolic switches, add an exciting twist to their versatile repertoire in bacterial pathogens. IMPORTANCE Regulatory small RNAs (sRNAs) of pathogens are coming to be recognized as highly important components of riboregulatory networks, involved in the control of essential cellular processes. They play a prominent role in adaptation to physiological changes as represented by different host environments. They can function as posttranscriptional regulators of gene expression to orchestrate metabolic adaptation to nutrient stresses. Here, we identified highly conserved sibling sRNAs in Neisseria meningitidis which are functionally involved in the regulation of gene expression of components of the tricarboxylic acid cycle. These novel sibling sRNAs that function by antisense mechanisms extend the so-called stringent response which connects metabolic status to colonization and possibly virulence as well as pathogenesis in meningococci.

............................................................................................................ 192 Introduction ....................................................................................................... 193 Materials & Methods ......................................................................................... 195 Bacterial strains and culture conditions ........................................................ 195 Plasmids and oligonucleotides ...................................................................... 197 Differential protein profiling using LC-MSE .................................................... 198 Construction of a meningococcal strain expressing a C-terminal 3XFLAGtagged cytochrome bc1 .................................................................................. 198 Liquid culture whole-cell fluorescence measurements and data processing 199 RNA isolation and RT-qPCR ............................................................................ 199 Western blotting ............................................................................................ 199 Results ............................................................................................................... 201 Proteomic analysis of nrrF mutant vs. overexpression strains show changes in proteins involved in iron homeostasis and oxidative stress response .......... 201 In silico target-interaction analysis of petA, a novel putative target of NrrF 203 Cytochrome bc1 levels are repressed upon expression of nrrF ..................... 203 Translational repression of a petA 5’UTR containing region fused to gfp upon expression of nrrF in E. coli ............................................................................ 206 NrrF represses PetABC by a direct interaction between NrrF and the 5’UTR of petABC ........................................................................................................... 207 Contribution of Hfq to post-transcriptional regulation of petA by NrrF ....... 208 Discussion .......................................................................................................... 209 Acknowledgements ........................................................................................... 213 Footnotes ........................................................................................................... 213 References ......................................................................................................... 214 Supplementary Data .......................................................................................... 220

NrrF is a small regulatory RNA of the human pathogen Neisseria meningitidis. NrrF was previously shown to repress succinate dehydrogenase (sdhCDAB) under control of the ferric uptake regulator (Fur). Here, we provide evidence that cytochrome bc1, encoded by the polycistronic mRNA petABC, is a NrrF target as well. We demonstrated differential expression of cytochrome bc1 comparing wild‐type meningococci and meningococci expressing NrrF when sufficient iron is available. Using a gfp‐reporter system monitoring translational control and target recognition of sRNA in Escherichia coli, we show that interaction between NrrF and the 5′ untranslated region of the petABC mRNA results in its repression. The NrrF region essential for repression of petABC was identified by site‐directed mutagenesis and is fully conserved among meningococci. Our results provide further insights into the mechanism by which Fur controls essential components of the N. meningitidis respiratory chain. Adaptation of cytochrome bc1 complex component levels upon iron limitation is post‐transcriptionally regulated via the small regulatory RNA NrrF.

Objectives: To determine the genotypes of serogroup Y meningococcus (MenY), and to determine the prevalence of and identify factors associated with MenY lpxL1 variants.Methods: Isolates, collected from 2003 to 2007 through national surveillance for invasive meningococcal disease, were characterized by multilocus sequence typing and screened for interleukin-6 induction. LpxL1 genes were sequenced from low IL-6 inducers.Results: MenY represented 13% (n = 219/1702) of meningococcal disease. Clonal complex (cc) 175, ST-23/Cluster A3 (cc23), cc11 and cc167 accounted for 82% (176/214), 11% (24/214), 3% (6/214) and 3% (7/214) respectively. Low cytokine induction was evident in 15% (32/218). Cc23 isolates (24/24) had an lpxL1 mutation, while among the remaining isolates the proportion of lpxL1 variants was 4% (8/189, p < 0.001), and these were all cc175. Compared to wild type isolates, lpxL1 variants were associated with patients aged 5-14 years [unadjusted OR (95% CI): 4.3 (1.5-12)] or 15-24 years [unadjusted OR (95% CI): 9.1 (2.8-29)] compared to children < 5 years; and were more likely have been isolated from CSF than blood [unadjusted OR (95% CI): 3.5 (1-11.9)]. On multivariable analysis, age remained significant [adjusted OR (95% CI), 5-14 years: 4.2 (1.5-12); 15-24 years: 8.9 (2.7-29)].Conclusion: LpxL1 variants were associated with cc23 among young adults. (C) 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

Objective: Lipopolysaccharide (LPS) is a major component of the Neisseria meningitidis outer membrane. Here we report a patient with meningococcal meningitis of which the causative isolate lacked LPS. Thus far, no naturally occurring LPS-deficient meningococcal isolate has been known to cause clinical disease.Methods: We used SDS-PAGE, silver staining and LPS-specific antibodies in whole cell ELISA to determine LPS presence in the causative isolate. Meningococcal whole genome sequencing was performed using Roche 454-sequencing. The N. meningitidis strain MC58 was used to compare all LPS biosynthesis associated genes. We compared growth characteristics of Escherichia coli transformed with a plasmid containing 2 lpxH types.Results: The patient presented with isolated thunderclap headache. Analysis of the causative N. meningitidis showed no LPS. Whole genome sequencing revealed a mutation located in lpxH explaining LPS-deficiency. Expression of this lpxH variant in E. coli resulted in growth impairment compared to E. coli expressing the meningococcal wild type lpxH variant. In addition, inactivating lpxH in N. meningitidis H44/76 by insertional inactivation with a kanamycin cassette resulted in a LPS-deficient phenotype.Conclusions: We describe invasive meningococcal disease caused by a naturally occurring LPS-deficient meningococcal isolate. (C) 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Background Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis , several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA -related gene cassettes, called tpsC , are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be. Results We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination. Conclusions Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.

BACKGROUND:Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their lpxL1 gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation.METHODS:A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The lpxL1 gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records.RESULTS:Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p = 0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p = 0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09-0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07-0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics.CONCLUSIONS:In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.

Biomaterial-associated infection (BAI) is a major problem in modern medicine, and is often caused by Staphylococcus epidermidis. We aimed to raise monoclonal antibodies (mAbs) against major surface protein antigens of S. epidermidis, and to assess their possible protective activity in experimental BAI. Mice were vaccinated with a cell wall protein preparation of S. epidermidis. A highly immunodominant antigen was identified as Accumulation-associated protein (Aap). Mabs against Aap and against surface-exposed lipoteichoic acid (LTA) were used for passive immunization of mice in experimental biomaterial-associated infection. Neither anti-Aap nor antiLTA mAbs showed protection. Either with or without antibodies, tissue surrounding the implants was more often culture positive than the implants themselves, but bacterial adherence to the implants was significantly increased in mice injected with anti-LTA. In vitro, anti-Aap and anti-LTA did show binding to S. epidermidis, but no opsonic activity was observed. We conclude that antibodies against S. epidermidis LTA or Aap showed no opsonic activity and did not protect mice against BAI. Moreover, the increase in binding to implanted biomaterial suggests that passive immunization may increase the risk for BAI.

Infections associated with implanted biomedical devices (BAI) are predominantly caused by Staphylococcus epidermidis. We previously observed in murine experimental BAI that S. epidermidis persists in peri-implant tissue rather than on the implanted biomaterial itself (Boelens et al., J Infect Dis 2000;181:1337-1349; Broekhuizen et al., Infect Immun 2007;75:1129-1136). To investigate the efficacy of rifampicin/vancomycin to clear S. epidermidis from implants and peri-implant tissues, mice with two implants were challenged with 10(7) cfu S. epidermidis per implant and received daily injections of rifampicin (25 mg/kg) and vancomycin (50 mg/kg). On the day of termination, implants and peri-implant tissue were collected and processed for culture and histology. After 1 and 8 days, implants of control mice were culture positive in 14/18 and 5/16 cases, respectively, and tissue biopsies were all culture positive. In the antibiotic-treated mice, bacteria were recovered from only 1/18 and 1/16 implants after 1 and 8 days, respectively, whereas the tissues were culture positive in 14/18 and 7/16 biopsies, respectively. In microscopy, bacteria were seen in the tissue at a distance of several cell layers from the tissue-implant interface, colocalized with host cells. Thus, although a regimen of rifampicin/vancomycin sterilized the implants, S. epidermidis persisted in peri-implant tissue, which might be an as yet unrecognized reservoir in the pathogenesis of BAI.

ABSTRACT Biomaterial-associated infections (BAI), which are predominantly caused by Staphylococcus epidermidis , are a significant problem in modern medicine. Biofilm formation is considered the pivotal element in the pathogenesis, but in previous mouse studies we retrieved S. epidermidis from peri-implant tissue. To assess the kinetics and generality of tissue colonization, we investigated BAI using two S. epidermidis strains, two biomaterials, and two mouse strains. With small inocula all implants were culture negative, whereas surrounding tissues were positive. When higher doses were used, tissues were culture positive more often than implants, with higher numbers of CFU. This was true for the different biomaterials tested, for both S. epidermidis strains, at different times, and for both mouse strains. S. epidermidis colocalized with host cells at a distance that was >10 cell layers from the biomaterial-tissue interface. We concluded that in mouse experimental BAI S. epidermidis peri-implant tissue colonization is more important than biofilm formation.

ABSTRACT Nucleotide sequencing is the standard molecular method for determination of the β-lactamase gene present in an isolate. Using minimal sequence quality values prevents misidentification of bla SHV genes, as illustrated by three strains of three different species that each contained two different bla SHV alleles, SHV-2 and SHV-12.