Anna de Breij

The Success of Acinetobacter Species; Genetic, Metabolic and Virulence Attributes

An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606T was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606T, A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606T and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species.

Do Biofilm Formation and Interactions with Human Cells Explain the Clinical Success of Acinetobacter baumannii

Background The dramatic increase in antibiotic resistance and the recent manifestation in war trauma patients underscore the threat of Acinetobacter baumannii as a nosocomial pathogen. Despite numerous reports documenting its epidemicity, little is known about the pathogenicity of A. baumannii. The aim of this study was to obtain insight into the factors that might explain the clinical success of A. baumannii. Methodology/Principal Findings We compared biofilm formation, adherence to and inflammatory cytokine induction by human cells for a large panel of well-described strains of A. baumannii and compared these features to that of other, clinically less relevant Acinetobacter species. Results revealed that biofilm formation and adherence to airway epithelial cells varied widely within the various species, but did not differ among the species. However, airway epithelial cells and cultured human macrophages produced significantly less inflammatory cytokines upon exposure to A. baumannii strains than to strains of A. junii, a species infrequently causing infection. Conclusion/Significance The induction of a weak inflammatory response may provide a clue to the persistence of A. baumannii in patients.

CsuA/BABCDE-dependent pili are not involved in the adherence of Acinetobacter baumannii ATCC19606T to human airway epithelial cells and their inflammatory response

Acinetobacter baumannii is a nosocomial pathogen responsible for outbreaks of infection worldwide. The factors associated with its ability to colonize/infect human hosts are largely unknown. Adherence to host cells is the first step in colonization/infection, which can be followed by biofilm formation. A. baumannii ATCC19606(T) biofilm formation on abiotic surfaces depends on expression of the CsuA/BABCDE chaperonee-usher pili assembly system. The present study focused on the involvement of CsuA/BABCDE-dependent pili in the interactions between A. baumannii 19606(T) and human bronchial epithelial cells and sheep erythrocytes. Light microscopy analysis revealed that CsuE-mutant #144 adhered to more bronchial epithelial cells than the parental strain. Similar amounts of interleukin (IL)-6 and IL-8 were produced by bronchial epithelial cells in response to these two bacterial strains. Scanning electron microscopy revealed the presence of two types of surface extensions on ATCC19606(T), i.e., short (29 nm; 5-140 nm) pili and long (260 nm; 143-1008 nm) extensions. The latter were not observed on the CsuE-mutant and therefore are likely the previously described CsuA/BABCDE-encoded extensions. We conclude that CsuA/BABCDE-dependent pili are not involved in adherence of A. baumannii ATCC19606(T) to bronchial epithelial cells. The structure of the short pili and their possible role in adherence to human cells requires further investigation.

LL-37-Derived Peptides Eradicate Multidrug-Resistant Staphylococcus aureus from Thermally Wounded Human Skin Equivalents

ABSTRACT Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria.

A doxycycline-loaded polymer-lipid encapsulation matrix coating for the prevention of implant-related osteomyelitis due to doxycycline-resistant methicillin-resistant Staphylococcus aureus.

Implant-associated bone infections caused by antibiotic-resistant pathogens pose significant clinical challenges to treating physicians. Prophylactic strategies that act against resistant organisms, such as methicillin-resistant Staphylococcus aureus (MRSA), are urgently required. In the present study, we investigated the efficacy of a biodegradable Polymer-Lipid Encapsulation MatriX (PLEX) loaded with the antibiotic doxycycline as a local prophylactic strategy against implant-associated osteomyelitis. Activity was tested against both a doxycycline-susceptible (doxy(S)) methicillin-susceptible S. aureus (MSSA) as well as a doxycycline-resistant (doxy(R)) methicillin-resistant S. aureus (MRSA). In vitro elution studies revealed that 25% of the doxycycline was released from the PLEX-coated implants within the first day, followed by a 3% release per day up to day 28. The released doxycycline was highly effective against doxy(S) MSSA for at least 14days in vitro. A bolus injection of doxycycline mimicking a one day release from the PLEX-coating reduced, but did not eliminate, mouse subcutaneous implant-associated infection (doxy(S) MSSA). In a rabbit intramedullary nail-related infection model, all rabbits receiving a PLEX-doxycycline-coated nail were culture negative in the doxy(S) MSSA-group and the surrounding bone displayed a normal physiological appearance in both histological sections and radiographs. In the doxy(R) MRSA inoculated rabbits, a statistically significant reduction in the number of culture-positive samples was observed for the PLEX-doxycycline-coated group when compared to the animals that had received an uncoated nail, although the reduction in bacterial burden did not reach statistical significance. In conclusion, the PLEX-doxycycline coating on titanium alloy implants provided complete protection against implant-associated MSSA osteomyelitis, and resulted in a significant reduction in the number of culture positive samples when challenged with a doxycycline-resistant MRSA.

Three-Dimensional Human Skin Equivalent as a Tool To Study Acinetobacter baumannii Colonization

ABSTRACT Acinetobacter baumannii can colonize body surfaces of hospitalized patients. From these sites, invasion into the host and spread to other patients and the hospital environment may occur. The eradication of the organism from the patients skin is an important infection control strategy during epidemic and endemic episodes. In this study, a three-dimensional (3D), air-exposed human epidermal skin equivalent was exploited to study Acinetobacter skin colonization. We characterized the adherence of A. baumannii ATCC 19606T and Acinetobacter junii RUH2228T to and biofilm formation on the skin equivalent and the responses to these bacteria. Furthermore, we assessed the ability of the disinfectant chlorhexidine to decolonize the skin equivalents. The results revealed that both strains replicated on the stratum corneum for up to 72 h but did not invade the epidermis. A. baumannii, in contrast to A. junii, formed large biofilms on the stratum corneum. Bacterial colonization did not affect keratinocyte activation, proliferation, or differentiation, nor did it induce a strong inflammatory response. Disinfection with chlorhexidine solution resulted in complete eradication of A. baumannii from the skin, without detrimental effects. This 3D model is a promising tool to study skin colonization and to evaluate the effects of novel disinfectant and antimicrobial strategies.

Lower Expression of TLR2 and SOCS-3 Is Associated with Schistosoma haematobium Infection and with Lower Risk for Allergic Reactivity in Children Living in a Rural Area in Ghana

Background Helminth infections are prevalent in rural areas of developing countries and have in some studies been negatively associated with allergic disorders and atopy. In this context little is known of the molecular mechanisms of modulation involved. We have characterized the innate immune responses, at the molecular level, in children according to their helminth infection status and their atopic reactivity to allergens. Methodology/Principal Findings The mRNA expression of several genes of the innate immune system that have been associated with microbial exposure and allergy was examined in 120 school children in a rural area in Ghana. Helminth infections were common and atopy rare in the study area. The analysis of gene expression in ex vivo whole blood samples reflected the levels of corresponding proteins. Using this approach in a population of school children in whom the presence of Schistosoma haematobium infection was associated with protection from atopic reactivity, we found that the level of TLR2 and SOCS-3, genes associated with atopy in the children, were significantly downregulated by presence of S. haematobium infection. Conclusions S. haematobium infections modulate the expression of genes of the innate immune system (TLR2 and SOCS-3); these are genes that are associated with increased allergic inflammatory processes, providing a molecular link between the negative association of this infection and atopy in rural children in Ghana.

Differences in Acinetobacter baumannii Strains and Host Innate Immune Response Determine Morbidity and Mortality in Experimental Pneumonia

Despite many reports documenting its epidemicity, little is known on the interaction of Acinetobacter baumannii with its host. To deepen our insight into this relationship, we studied persistence of and host response to different A. baumannii strains including representatives of the European (EU) clones I–III in a mouse pneumonia model. Neutropenic mice were inoculated intratracheally with five A. baumannii strains and an A. junii strain and at several days morbidity, mortality, bacterial counts, airway inflammation, and chemo- and cytokine production in lungs and blood were determined. A. baumannii RUH875 and RUH134 (EU clone I and II, respectively) and sporadic strain LUH8326 resulted in high morbidity/mortality, whereas A. baumannii LUH5875 (EU clone III, which is less widespread than clone I and II) caused less symptoms. A. baumannii type strain RUH3023T and A. junii LUH5851 did not cause disease. All strains, except A. baumannii RUH3023T and A. junii LUH5851, survived and multiplied in the lungs for several days. Morbidity and mortality were associated with the severity of lung pathology and a specific immune response characterized by low levels of anti-inflammatory (IL-10) and specific pro-inflammatory (IL-12p40 and IL-23) cytokines at the first day of infection. Altogether, a striking difference in behaviour among the A. baumannii strains was observed with the clone I and II strains being most virulent, whereas the A. baumannii type strain, which is frequently used in virulence studies appeared harmless.

Inflammatory and Antimicrobial Responses to Methicillin-Resistant Staphylococcus aureus in an In Vitro Wound Infection Model

Treatment of patients with burn wound infections may become complicated by the presence of antibiotic resistant bacteria and biofilms. Herein, we demonstrate an in vitro thermal wound infection model using human skin equivalents (HSE) and biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) for the testing of agents to combat such infections. Application of a liquid nitrogen-cooled metal device on HSE produced reproducible wounds characterized by keratinocyte death, detachment of the epidermal layer from the dermis, and re-epithelialization. Thermal wounding was accompanied by up-regulation of markers for keratinocyte activation, inflammation, and antimicrobial responses. Exposure of thermal wounded HSEs to MRSA resulted in significant numbers of adherent MRSA/HSE after 1 hour, and multiplication of these bacteria over 24-48 hours. Exposure to MRSA enhanced expression of inflammatory mediators such as TLR2 (but not TLR3), IL-6 and IL-8, and antimicrobial proteins human β-defensin-2, -3 and RNAse7 by thermal wounded as compared to control HSEs. Moreover, locally applied mupirocin effectively reduced MRSA counts on (thermal wounded) HSEs by more than 99.9% within 24 hours. Together, these data indicate that this thermal wound infection model is a promising tool to study the initial phase of wound colonization and infection, and to assess local effects of candidate antimicrobial agents.

Phospholipid-driven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems.

OP-145, a synthetic antimicrobial peptide developed from a screen of the human cathelicidin LL-37, displays strong antibacterial activities and is--at considerably higher concentrations--lytic to human cells. To obtain more insight into its actions, we investigated the interactions between OP-145 and liposomes composed of phosphatidylglycerol (PG) and phosphatidylcholine (PC), resembling bacterial and mammalian membranes, respectively. Circular dichroism analyses of OP-145 demonstrated a predominant α-helical conformation in the presence of both membrane mimics, indicating that the different membrane-perturbation mechanisms are not due to different secondary structures. Membrane thinning and formation of quasi-interdigitated lipid-peptide structures was observed in PG bilayers, while OP-145 led to disintegration of PC liposomes into disk-like micelles and bilayer sheets. Although OP-145 was capable of binding lipoteichoic acid and peptidoglycan, the presence of these bacterial cell wall components did not retain OP-145 and hence did not interfere with the activity of the peptide toward PG membranes. Furthermore, physiological Ca++ concentrations did neither influence the membrane activity of OP-145 in model systems nor the killing of Staphylococcus aureus. However, addition of OP-145 at physiological Ca++-concentrations to PG membranes, but not PC membranes, resulted in the formation of elongated enrolled structures similar to cochleate-like structures. In summary, phospholipid-driven differences in incorporation of OP-145 into the lipid bilayers govern the membrane activity of the peptide on bacterial and mammalian membrane mimics.