Characterization of Acinetobacter baumannii Copper Resistance Reveals a Role in Virulence.

Acinetobacter baumannii is often highly drug-resistant and causes severe infections in compromised patients. These infections can be life threatening due to limited treatment options. Copper is inherently antimicrobial and increasing evidence indicates that copper containing formulations may serve as non-traditional therapeutics against multidrug-resistant bacteria. We previously reported that A. baumannii is sensitive to high concentrations of copper. To understand A. baumannii copper resistance at the molecular level, herein we identified putative copper resistance components and characterized 21 strains bearing mutations in these genes. Eight of the strains displayed a copper sensitive phenotype (pcoA, pcoB, copB, copA/cueO, copR/cusR, copS/cusS, copC, copD); the putative functions of these proteins include copper transport, oxidation, sequestration, and regulation. Importantly, many of these mutant strains still showed increased sensitivity to copper while in a biofilm. Inductively coupled plasma mass spectrometry revealed that many of these strains had defects in copper mobilization, as the mutant strains accumulated more intracellular copper than the wild-type strain. Given the crucial antimicrobial role of copper-mediated killing employed by the immune system, virulence of these mutant strains was investigated in Galleria mellonella; many of the mutant strains were attenuated. Finally, the cusR and copD strains were also investigated in the murine pneumonia model; both were found to be important for full virulence. Thus, copper possesses antimicrobial activity against multidrug-resistant A. baumannii, and copper sensitivity is further increased when copper homeostasis mechanisms are interrupted. Importantly, these proteins are crucial for full virulence of A. baumannii and may represent novel drug targets.

American Civil War plant medicines inhibit growth, biofilm formation, and quorum sensing by multidrug-resistant bacteria.

A shortage of conventional medicine during the American Civil War (1861-1865) spurred Confederate physicians to use preparations of native plants as medicines. In 1863, botanist Francis Porcher compiled a book of medicinal plants native to the southern United States, including plants used in Native American traditional medicine. In this study, we consulted Porcher's book and collected samples from three species that were indicated for the formulation of antiseptics: Liriodendron tulipifera, Aralia spinosa, and Quercus alba. Extracts of these species were tested for the ability to inhibit growth in three species of multidrug-resistant pathogenic bacteria associated with wound infections: Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii. Extracts were also tested for biofilm and quorum sensing inhibition against S. aureus. Q. alba extracts inhibited growth in all three species of bacteria (IC50 64, 32, and 32 µg/mL, respectively), and inhibited biofilm formation (IC50 1 µg/mL) in S. aureus. L. tulipifera extracts inhibited biofilm formation (IC50 32 µg/mL) in S. aureus. A. spinosa extracts inhibited biofilm formation (IC50 2 µg/mL) and quorum sensing (IC50 8 µg/mL) in S. aureus. These results support that this selection of plants exhibited some antiseptic properties in the prevention and management of wound infections during the conflict.

Streptococcus pneumoniae Modulates Staphylococcus aureus Biofilm Dispersion and the Transition from Colonization to Invasive Disease.

Streptococcus pneumoniae and Staphylococcus aureus are ubiquitous upper respiratory opportunistic pathogens. Individually, these Gram-positive microbes are two of the most common causative agents of secondary bacterial pneumonia following influenza A virus infection, and they constitute a significant source of morbidity and mortality. Since the introduction of the pneumococcal conjugate vaccine, rates of cocolonization with both of these bacterial species have increased, despite the traditional view that they are antagonistic and mutually exclusive. The interactions between S. pneumoniae and S. aureus in the context of colonization and the transition to invasive disease have not been characterized. In this report, we show that S. pneumoniae and S. aureus form stable dual-species biofilms on epithelial cells in vitro When these biofilms are exposed to physiological changes associated with viral infection, S. pneumoniae disperses from the biofilm, whereas S. aureus dispersal is inhibited. These findings were supported by results of an in vivo study in which we used a novel mouse cocolonization model. In these experiments, mice cocolonized in the nares with both bacterial species were subsequently infected with influenza A virus. The coinfected mice almost exclusively developed pneumococcal pneumonia. These results indicate that despite our previous report that S. aureus disseminates into the lungs of mice stably colonized with these bacteria following influenza A virus infection, cocolonization with S. pneumoniae in vitro and in vivo inhibits S. aureus dispersal and transition to disease. This study provides novel insight into both the interactions between S. pneumoniae and S. aureus during carriage and the transition from colonization to secondary bacterial pneumonia.IMPORTANCE In this study, we demonstrate that Streptococcus pneumoniae can modulate the pathogenic potential of Staphylococcus aureus in a model of secondary bacterial pneumonia. We report that host physiological signals related to viral infection cease to elicit a dispersal response from S. aureus while in a dual-species setting with S. pneumoniae, in direct contrast to results of previous studies with each species individually. This study underscores the importance of studying polymicrobial communities and their implications in disease states.

Host Physiologic Changes Induced by Influenza A Virus Lead to Staphylococcus aureus Biofilm Dispersion and Transition from Asymptomatic Colonization to Invasive Disease.

UNLABELLED: Staphylococcus aureus is a ubiquitous opportunistic human pathogen and a major health concern worldwide, causing a wide variety of diseases from mild skin infections to systemic disease. S. aureus is a major source of severe secondary bacterial pneumonia after influenza A virus infection, which causes widespread morbidity and mortality. While the phenomenon of secondary bacterial pneumonia is well established, the mechanisms behind the transition from asymptomatic colonization to invasive staphylococcal disease following viral infection remains unknown. In this report, we have shown that S. aureus biofilms, grown on an upper respiratory epithelial substratum, disperse in response to host physiologic changes related to viral infection, such as febrile range temperatures, exogenous ATP, norepinephrine, and increased glucose. Mice that were colonized with S. aureus and subsequently exposed to these physiologic stimuli or influenza A virus coinfection developed pronounced pneumonia. This study provides novel insight into the transition from colonization to invasive disease, providing a better understanding of the events involved in the pathogenesis of secondary staphylococcal pneumonia. IMPORTANCE: In this study, we have determined that host physiologic changes related to influenza A virus infection causes S. aureus to disperse from a biofilm state. Additionally, we report that these same host physiologic changes promote S. aureus dissemination from the nasal tissue to the lungs in an animal model. Furthermore, this study identifies important aspects involved in the transition of S. aureus from asymptomatic colonization to pneumonia.

Zinc protects against Shiga-toxigenic Escherichia coli by acting on host tissues as well as on bacteria.

BACKGROUND: Zinc supplements can treat or prevent enteric infections and diarrheal disease. Many articles on zinc in bacteria, however, highlight the essential nature of this metal for bacterial growth and virulence, suggesting that zinc should make infections worse, not better. To address this paradox, we tested whether zinc might have protective effects on intestinal epithelium as well as on the pathogen. RESULTS: Using polarized monolayers of T84 cells we found that zinc protected against damage induced by hydrogen peroxide, as measured by trans-epithelial electrical resistance. Zinc also reduced peroxide-induced translocation of Shiga toxin (Stx) across T84 monolayers from the apical to basolateral side. Zinc was superior to other divalent metals to (iron, manganese, and nickel) in protecting against peroxide-induced epithelial damage, while copper also showed a protective effect.The SOS bacterial stress response pathway is a powerful regulator of Stx production in STEC. We examined whether zinc's known inhibitory effects on Stx might be mediated by blocking the SOS response. Zinc reduced expression of recA, a reliable marker of the SOS. Zinc was more potent and more efficacious than other metals tested in inhibiting recA expression induced by hydrogen peroxide, xanthine oxidase, or the antibiotic ciprofloxacin. The close correlation between zinc's effects on recA/SOS and on Stx suggested that inhibition of the SOS response is one mechanism by which zinc protects against STEC infection. CONCLUSIONS: Zinc's ability to protect against enteric bacterial pathogens may be the result of its combined effects on host tissues as well as inhibition of virulence in some pathogens. Research focused solely on the effects of zinc on pathogenic microbes may give an incomplete picture by failing to account for protective effects of zinc on host epithelia.

Biofilm formation enhances fomite survival of Streptococcus pneumoniae and Streptococcus pyogenes.

Both Streptococcus pyogenes and Streptococcus pneumoniae are widely thought to rapidly die outside the human host, losing infectivity following desiccation in the environment. However, to date, all literature investigating the infectivity of desiccated streptococci has used broth-grown, planktonic populations. In this study, we examined the impact of biofilm formation on environmental survival of clinical and laboratory isolates of S. pyogenes and S. pneumoniae as both organisms are thought to colonize the human host as biofilms. Results clearly demonstrate that while planktonic cells that are desiccated rapidly lose viability both on hands and abiotic surfaces, such as plastic, biofilm bacteria remain viable over extended periods of time outside the host and remain infectious in a murine colonization model. To explore the level and extent of streptococcal fomite contamination that children might be exposed to naturally, direct bacteriologic cultures of items in a day care center were conducted, which demonstrated high levels of viable streptococci of both species. These findings raise the possibility that streptococci may survive in the environment and be transferred from person to person via fomites contaminated with oropharyngeal secretions containing biofilm streptococci.

High levels of genetic recombination during nasopharyngeal carriage and biofilm formation in Streptococcus pneumoniae.

UNLABELLED: Transformation of genetic material between bacteria was first observed in the 1920s using Streptococcus pneumoniae as a model organism. Since then, the mechanism of competence induction and transformation has been well characterized, mainly using planktonic bacteria or septic infection models. However, epidemiological evidence suggests that genetic exchange occurs primarily during pneumococcal nasopharyngeal carriage, which we have recently shown is associated with biofilm growth, and is associated with cocolonization with multiple strains. However, no studies to date have comprehensively investigated genetic exchange during cocolonization in vitro and in vivo or the role of the nasopharyngeal environment in these processes. In this study, we show that genetic exchange during dual-strain carriage in vivo is extremely efficient (10(-2)) and approximately 10,000,000-fold higher than that measured during septic infection (10(-9)). This high transformation efficiency was associated with environmental conditions exclusive to the nasopharynx, including the lower temperature of the nasopharynx (32 to 34°C), limited nutrient availability, and interactions with epithelial cells, which were modeled in a novel biofilm model in vitro that showed similarly high transformation efficiencies. The nasopharyngeal environmental factors, combined, were critical for biofilm formation and induced constitutive upregulation of competence genes and downregulation of capsule that promoted transformation. In addition, we show that dual-strain carriage in vivo and biofilms formed in vitro can be transformed during colonization to increase their pneumococcal fitness and also, importantly, that bacteria with lower colonization ability can be protected by strains with higher colonization efficiency, a process unrelated to genetic exchange. IMPORTANCE: Although genetic exchange between pneumococcal strains is known to occur primarily during colonization of the nasopharynx and colonization is associated with biofilm growth, this is the first study to comprehensively investigate transformation in this environment and to analyze the role of environmental and bacterial factors in this process. We show that transformation efficiency during cocolonization by multiple strains is very high (around 10(-2)). Furthermore, we provide novel evidence that specific aspects of the nasopharyngeal environment, including lower temperature, limited nutrient availability, and epithelial cell interaction, are critical for optimal biofilm formation and transformation efficiency and result in bacterial protein expression changes that promote transformation and fitness of colonization-deficient strains. The results suggest that cocolonization in biofilm communities may have important clinical consequences by facilitating the spread of antibiotic resistance and enabling serotype switching and vaccine escape as well as protecting and retaining poorly colonizing strains in the pneumococcal strain pool.