Llinos G. Harris

Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

ABSTRACT Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

Analysis of Ebh, a 1.1-Megadalton Cell Wall-Associated Fibronectin-Binding Protein of Staphylococcus aureus

ABSTRACT In order for Staphylococcus aureus to adhere to host extracellular matrix (ECM) substrates, it elicits a wide range of surface proteins. We have characterized a novel ∼1.1-MDa protein in S. aureus, termed Ebh (for ECM-binding protein homologue), which has homology to other ECM-binding proteins. Ebh consists of several domains, including a large central region with 44 imperfect repeats of 126 amino acids. Expression analysis revealed ebh to be growth phase regulated and repressed by agr. A fragment of the central repeat region of Ebh was cloned, overexpressed, and used in ligand-binding studies to determine Ebh function. The recombinant protein was found to specifically bind human fibronectin. Ebh is produced during human infection since serum samples taken from patients with confirmed S. aureus infections were found to contain anti-Ebh antibodies. Localization studies revealed Ebh to be cell envelope associated and is proposed to form a specialized surface structure involved in cellular adhesion.

Disruption of Staphylococcus Epidermidis Biofilms by Medicinal Maggot Lucilia Sericata Excretions/Secretions:

Chronic infections are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. With the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduced for the treatment of chronic wounds. Studies have shown that the excretion/secretions (ES) of Lucilia sericata larvae (maggots) contain many bioactive compounds which may contribute to the efficacy of maggot therapy. The present study evaluates the effect of L. sericata ES on the formation and disruption of S. epidermidis 7457 and 5179-R1 biofilms. These strains employ either polysaccharide intercellular adhesin (PIA) or accumulation associated protein (Aap) for intercellular adhesion. A semiquantitative biofilm assay was used to measure the formation/disruption of S. epidermidis 7457 and 5179-R1 biofilms by ES. ES activity was characterized according to concentration, incubation time and temperature, thermal stability, and size. Immunofluorescence microscopy was used to ascertain the effect of ES on PIA and Aap. In the presence of ES, S. epidermidis 7457 and 5179-R1 nascent bio film formation was inhibited, and pre-formed biofilms disrupted. ES activity was temperature and time dependent, inactivated by heat treatment, and disruption depended on the mechanism of intercellular adhesion. The molecule(s) responsible was >10 kDa in size and appeared to have protease or glucosaminidase activity. ES interferes with S. epidermidis biofilm formation, specifically degrading factors employed in biofilm accumulation, which would increase bacterial susceptibility to antibiotics and the hosts immune system. In purified form, ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci.

Ecological Overlap and Horizontal Gene Transfer in Staphylococcus aureus and Staphylococcus epidermidis

The opportunistic pathogens Staphylococcus aureus and Staphylococcus epidermidis represent major causes of severe nosocomial infection, and are associated with high levels of mortality and morbidity worldwide. These species are both common commensals on the human skin and in the nasal pharynx, but are genetically distinct, differing at 24% average nucleotide divergence in 1,478 core genes. To better understand the genome dynamics of these ecologically similar staphylococcal species, we carried out a comparative analysis of 324 S. aureus and S. epidermidis genomes, including 83 novel S. epidermidis sequences. A reference pan-genome approach and whole genome multilocus-sequence typing revealed that around half of the genome was shared between the species. Based on a BratNextGen analysis, homologous recombination was found to have impacted on 40% of the core genes in S. epidermidis, but on only 24% of the core genes in S. aureus. Homologous recombination between the species is rare, with a maximum of nine gene alleles shared between any two S. epidermidis and S. aureus isolates. In contrast, there was considerable interspecies admixture of mobile elements, in particular genes associated with the SaPIn1 pathogenicity island, metal detoxification, and the methicillin-resistance island SCCmec. Our data and analysis provide a context for considering the nature of recombinational boundaries between S. aureus and S. epidermidis and, the selective forces that influence realized recombination between these species.

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement.

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.

Rapid differentiation of Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci and meticillin susceptibility testing directly from growth-positive blood cultures by multiplex real-time PCR

This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE genes for rapid identification of Staphylococcus aureus, Staphylococcus epidermidis and non-S. epidermidis coagulase-negative staphylococci (CoNS), and meticillin susceptibility testing directly in positive blood cultures that grew Gram-positive cocci in clusters. A total of 100 positive blood cultures produced: 39 S. aureus [12 meticillin-resistant S. aureus (MRSA), 31% of all the S. aureus]; 30 S. epidermidis (56.6% of the CoNS), 8 Staphylococcus capitis (15.1%), 3 Staphylococcus saprophyticus (5.7%), 4 Staphylococcus hominis (7.5%), 3 Staphylococcus haemolyticus (5.7%), 2 Staphylococcus warneri (3.8%), 1 Staphylococcus cohnii (1.9%) and 2 unidentified Staphylococcus spp. (3.8%); and 1 Micrococcus luteus in pure culture. Two blood cultures had no growth on subculture and five blood cultures grew mixed CoNS. For the 95 blood cultures with pure growth or no growth on subculture, there was very good agreement between real-time PCR and the BD Phoenix identification system for staphylococcal species categorization in S. aureus, S. epidermidis and non-S. epidermidis CoNS and meticillin-resistance determination (Cohens unweighted kappa coefficient κ=0.882). All MRSA and meticillin-susceptible S. aureus were correctly identified by mecA amplification. PCR amplification of mecA was more sensitive for direct detection of meticillin-resistant CoNS in positive blood cultures than testing with the BD Phoenix system. There were no major errors when identifying staphylococcal isolates and their meticillin susceptibility within 2.5 h. Further studies are needed to evaluate the clinical benefit of using such a rapid test on the consumption of glycopeptide antibiotics and the alteration of empiric therapy in the situation of positive blood cultures growing staphylococci, and the respective clinical outcomes.

Comparison of bacterial identification by MALDI-TOF mass spectrometry and conventional diagnostic microbiology methods: agreement, speed and cost implications

ABSTRACT Identification of microbial pathogens still relies primarily on culture and phenotypic methods, which is labour-intensive and time-consuming. In this study, identification of bacteria with valid standard identification using BD Phoenix, API panels and other recommended procedures is compared to identification with matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry using the MALDI Biotyper (Bruker Daltonics) in the setting of a routine NHS diagnostic microbiology laboratory. In total, 928 bacterial isolates obtained from blood (n=463), wounds and pus (n=208), respiratory tract (n = 100), faeces (n=86) and urines (n=71) were analysed. There were 721 (77.7%) isolates with a MALDI Biotyper score ≥2.0, indicating secure genus and probable species identification; and 149 (16.1%) isolates with a score ≥1.7 and <2.0 indicating probable genus identification. The isolates with scores of ≥2.0 and ≥1.7 comprised 31 and 33 genera and 65 and 67 species, respectively. Overall, 99.4% and 99.1% of organism identifications were in agreement between the MALDI Biotyper and conventional identification at the genus level, and 89.3% and 87.8% at species level when analysing organisms with MALDI Biotyper scores ≥2.0 and ≥1.7, respectively. With many but not all organisms, identification at the genus level is sufficient; however, MALDI Biotyper separation of 208 staphylococci into Staphylococcus aureus and coagulase-negative staphylococci was always correct when scores were ≥1.7. First results were obtained after 5–10 min and analysis of a full 96-well target plate was completed in approximately 90 min. Substantial savings of between £1.79 and £2.56 per isolate, depending on the cost model of acquisition of the MALDI Biotyper system and number of isolates tested, would be realised when all 928 isolates were identified using the MALDI Biotyper and disk-susceptibility testing when compared to the cost for 618 Phoenix ID panels and 158 API panels and disk-susceptibility tests only (i.e., not taking into account costs incurred for identification of the remaining 152 mixed isolates). Microbial identification by MALDI Biotyper offers a rare opportunity for significant cost-neutral or even cost-saving quality improvements in medical diagnostics.

Identification of Clinical Isolates of α-Hemolytic Streptococci by 16S rRNA Gene Sequencing, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using MALDI Biotyper, and Conventional Phenotypic Methods: a Comparison

ABSTRACT Fifty-six α-hemolytic streptococcal isolates were identified using MALDI Biotyper MS (Bruker Daltonics), API 20 Strep (bioMérieux), and BD Phoenix (Becton, Dickinson). The gold standard for identification was 16S rRNA gene sequence analysis with 16S-23S rRNA intergenic spacer sequencing. The following percentages of isolates were correctly identified to the species level: MALDI Biotyper, 46%; BD Phoenix, 35%; and API 20 Strep, 26%.

Staphylococcus epidermidis in biomaterial-associated infections

Coagulase-negative staphylococci, mainly Staphylococcus epidermidis, are currently the most frequent cause of hospital acquired infections in the USA. Mostly, but not exclusively, S. epidermidis infections are linked to the use of implanted medical devices like central venous catheters, prosthetic joints and heart valves, pacemakers, cardiac assist devices, cerebrospinal fluid shunts, and intraocular lenses. As new molecular techniques reveal that S. epidermidis are by no means the most prominent bacteria of the skin and mucous membrane flora, the implication is that S. epidermidis has specific virulence factors, which transforms this commensal bacterial species into one of the most successful pathogens in modern medicine. A vast array of specific attachment factors for native and host protein-modified device surfaces and the ability to accumulate in adherent multilayered biofilms appear to be vital for the success of S. epidermidis as a pathogen. Biofilm formation contributes to the ability of the organism to withstand the host’s innate and acquired immune defense mechanisms and to resist antimicrobial therapy, so that device removal is a regular feature for the treatment of S. epidermidis biomaterial-associated infection. Recent developments in the understanding of S. epidermidis virulence are reviewed in this chapter.

Lucilia sericata chymotrypsin disrupts protein adhesin-mediated staphylococcal biofilm formation.

ABSTRACT Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.