First Report of Antimicrobial Susceptibility and Virulence Gene Characterization Associated with Staphylococcus aureus Carriage in Healthy Camels from Tunisia

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin; lukE–lukD, encoding bicomponent leukotoxin LukE–LukD; the clfB gene, encoding clumping factor B; the laminin gene, encoding laminin-binding protein; and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Transcriptomic analysis of human brain microvascular endothelial cells exposed to laminin binding protein (adhesion lipoprotein) and Streptococcus pneumoniae

Streptococcus pneumoniae invades the CNS and triggers a strong cellular response. To date, signaling events that occur in the human brain microvascular endothelial cells (hBMECs), in response to pneumococci or its surface adhesins are not mapped comprehensively. We evaluated the response of hBMECs to the adhesion lipoprotein (a laminin binding protein—Lbp) or live pneumococci. Lbp is a surface adhesin recently identified as a potential ligand, which binds to the hBMECs. Transcriptomic analysis was performed by RNA-seq of three independent biological replicates and validated with qRT-PCR using 11 genes. In total 350 differentially expressed genes (DEGs) were identified after infection with S. pneumoniae, whereas 443 DEGs when challenged with Lbp. Total 231 DEGs were common in both treatments. Integrative functional analysis revealed participation of DEGs in cytokine, chemokine, TNF signaling pathways and phagosome formation. Moreover, Lbp induced cell senescence and breakdown, and remodeling of ECM. This is the first report which maps complete picture of cell signaling events in the hBMECs triggered against S. pneumoniae and Lbp. The data obtained here could contribute in a better understanding of the invasion of pneumococci across BBB and underscores role of Lbp adhesin in evoking the gene expression in neurovascular unit.

Comparison of the Prevalence of Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) among Staphylococcus aureus Isolates in a Burn Unit with Non-Burning Units

Background: Staphylococcus aureus (S. aureus) is one of the most important pathogens in burn infections colonized in the nose and increase the risk of infections. Methods: Overall, 85 S. aureus isolates were isolated from clinical and nasal hospitalized patients and health care workers (HCWs) in a burn unit and non-burn units in Isfahan from June 2016 and September 2016. Genes encoding penicillin-binding protein 2a (mecA) and adhesive surface proteins, including fibronectin-binding proteins (fnbA, fnbB), fibrinogen binding protein (fib), laminin-binding protein(eno), collagen binding protein (cna), elastin binding protein (ebps), intracellular adhesion operon (icaA and icaD) were detected using PCR method. Results: The rate of methicillin-resistant S. aureus (MRSA) among burn and non-burn isolates were 62% (18/29) and 25% (14/56), respectively. The most prevalent MSCRAMMs genes in burn units were eno (86%) and fib (66%). The most common gene pattern in burn center was icaA+fib+eno. The frequency of icaD, fib and ebpS was higher in clinical samples than nasal samples. No relation was found between the MSCRAMMs genes in the burn unit and non-burn units. Conclusion: The high prevalence of MRSA in burn center can be a new challenge for clinicians. The higher frequency of icaD, fib and ebpS in clinical isolates than nasal isolates may reflect the important role of these genes in colonization and pathogenesis of S. aureus.

Effect of trans-Cinnamaldehyde on Methicillin-Resistant Staphylococcus aureus Biofilm Formation: Metabolic Activity Assessment and Analysis of the Biofilm-Associated Genes Expression

The effects of trans-cinnamaldehyde (TC) on transcriptional profiles of biofilm-associated genes and the metabolic activity of two methicillin-resistant Staphylococcus aureus (MRSA) strains showing a different degree of adherence to polystyrene, were evaluated. Metabolic activity of S. aureus in biofilm was significantly decreased in the presence of TC at 1/2 minimum biofilm inhibition concentration (MBIC). Expression levels of the genes encoding laminin binding protein (eno), elastin binding protein (ebps) and fibrinogen binding protein (fib) in the presence of TC at 1/2 MBIC were lower than in untreated biofilm in both the weakly and strongly adhering strain. The highest decrease of expression level was observed in case of fib in the strongly adhering strain, in which the amount of fib transcript was 10-fold lower compared to biofilm without TC. In the presence of TC at 1/2 MBIC after 3, 6, 8 and 12 h, the expression level of icaA and icaD, that are involved in the biosynthesis of polysaccharide intercellular adhesin, was above half lower in the weakly adhering strain compared to biofilm without TC. In the strongly adhering strain the highest decrease in expression of these genes was observed after 3 and 6 h. This study showed that TC is a promising anti-biofilm agent for use in MRSA biofilm-related infections.

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

Expression of the Biofilm-Associated Genes in Methicillin-Resistant Staphylococcus aureus in Biofilm and Planktonic Conditions

The role of genes that are essential for development of Staphylococcus aureus biofilm during infection is not fully known. mRNA from two methicillin-resistant S. aureus strains that formed weak and strong biofilm on polystyrene plates were isolated at five time points from cells grown in biofilm and planktonic culture. Quantitative real-time PCR analysis showed that the expression levels of investigated genes under biofilm conditions were significantly higher than under planktonic conditions. The expression levels of the gene encoding elastin binding protein (ebps) and laminin binding protein (eno) were significantly increased in biofilm at 3 h, both in strongly and weakly adhering strain. The peak expression of fib gene encoding fibrinogen binding protein was found at 6 and 8 h in the case of strongly and weakly adhering strain, respectively. The expression of icaA and icaD genes in both strains was significantly higher under biofilm conditions when comparing to planktonic cells during 12 h. The expression level of the genes encoding binding proteins and the glucosamine polymer polysaccharide intercellular adhesin (PIA) slowly decreased after 24 h. Finally, we found that the expression levels of genes encoding binding factors in weakly adhering strain were significantly lower than in strongly adhering strain.