Analysis of Acinetobacter baumannii survival in liquid media and on solid matrices as well as effect of disinfectants.

BACKGROUND: Acinetobacter baumannii is a cause of healthcare-associated infections and has considerable potential to survive on inanimate hospital surfaces under hostile conditions (e.g. disinfection or desiccation). AIM: To learn more about its survival strategy and capacity to persist in liquid media and on surfaces mimicking hospital environments. METHODS: The effect of temperature, nutrient deprivation, permanence on inanimate surfaces, and exposure to disinfectants on the survival of four A. baumannii strains (ATCC 19606T and three clinical isolates) was studied by monitoring the number of total and viable cells using fluorescent microscopy and of culturable cells by standard cultures. FINDINGS: Bacterial survival was differentially affected by temperature (cells maintained at 20°C remained culturable at least within 30 days) and physical environment (desiccation favoured cell resistance to stress at 37°C). Moreover, persistence was associated with two adaptation patterns: one linked to entry into the viable but non-culturable state, whereas the other apparently followed a bust-and-boom model. During a study on the effect of disinfectant (commercial bleach and quaternary ammonium compounds), it was found that treatment with these antibacterial compounds did not eliminate A. baumannii populations and provoked the reduction of culturable populations, although a fraction of cells remained culturable. CONCLUSION: The ability to persist for long periods on different surfaces, mimicking those usually found in hospitals, along with A. baumannii's capacity to survive after a disinfection process may account for the recurrent outbreaks in intensive care units.

Acinetobacter pittii biofilm formation on inanimate surfaces after long-term desiccation.

BACKGROUND: The survival of pathogenic micro-organisms in the healthcare environment has a major role in nosocomial infections. Among the responsible mechanisms enabling nosocomial pathogens to persist with these stress conditions is their ability to resist desiccation and to form biofilms. AIM: To investigate the survival behaviour of Acinetobacter pittii isolates on inert surfaces and saline microcosms. METHODS: Five A. pittii clinical strains were spotted over white laboratory coat fragments, glass, and plastic surfaces, or inoculated into sterile saline and monitored at room temperature for a period of 43 days. FINDINGS: Although the permanence on solid surfaces negatively affected the culturability of the strains used, a fraction of stressed cells survived for at least the period of study. On average, A. pittii culturability was reduced by 77.3%, 80.9%, and 68.1% in white coat, plastic, and glass surfaces, respectively. However, ∼85.6% of the populations retain their culturability in saline solution. Culturability correlated with the presence of cells with an intact membrane, as demonstrated after live/dead staining. Supplementation of the culture medium with sodium pyruvate favoured the culturability of strains from all conditions; but, in general, A. pittii populations did not enter a viable but non-culturable state. CONCLUSION: After long-term desiccation, all A. pittii strains retained, or even increased, their ability to form biofilms after they had been fed with nutrient media. This suggests that A. pittii may recover easily from desiccation and may express adherence factors to infect new hosts after rehydration.

Stenotrophomonas maltophilia healthcare-associated infections: identification of two main pathogenic genetic backgrounds.

BACKGROUND: Stenotrophomonas maltophilia is an opportunistic multi-drug-resistant bacterium responsible for healthcare-associated infections. Strategies for in-hospital infection control and management of carriers and environmental reservoirs remain controversial. AIM: To determine the population structure of S. maltophilia strains in hospitalized infected patients and to identify putative highly pathogenic subpopulations that require upgraded infection control measures. METHODS: Eighty-three diverse human strains of various clinical origins from 18 geographically distant hospitals were characterized phenotypically and genotypically using a multi-locus sequence typing (MLST) approach. FINDINGS: Neither a predominant nor emerging sequence type (ST) was identified. Among the 80 typeable strains, only 29% corresponded to described STs, especially ST5 (N=6) and ST4/26/31 (N=2). The ST distribution and the phylogenic tree based on the concatenated MLST genes did not account for geographical, clinical origin or antimicrobial susceptibility clustering. A phylogenic tree that included 173 ST profiles from the MLST database and the 80 typeable strains confirmed the high genetic diversity of S. maltophilia, the previously reported genogroup organization and the predominance of genogroup 6, as it represented 41% (33/80) of the strains. Unexpectedly, genogroup 2 was the second most prevalent genogroup and included 16% (13/80) of the strains. These genogroups represented 57% (20/35) of the strains in respiratory patients and 75% (9/12) of the strains in patients with cystic fibrosis. CONCLUSION: Beyond MLST, the over-representation of some genogroups among strains responsible for healthcare-associated infections was confirmed. Genogrouping affiliation is recommended to implement infection control measures selectively for the most pathogenic strains isolated from patient or environmental reservoirs.

Immunization of sea bream (Sparus aurata) juveniles against Photobacterium damselae subsp. piscicida by short bath: Effect on some pro-inflammatory molecules and the Mx gene expression.

Cytokines are a family of proteins derived from macrophages, lymphocytes, granulocytes, mast cells and epithelial cells and can be divided into interferons (IFNs), Interleukins (ILs) and Tumor Necrosis factors (TNFs) among others. The presence of cytokines in a wide number of fish species has been proved and several molecules types have been already cloned and sequenced. In this work some proinflamatory molecules and Mx gene were detected in the liver of vaccinated sea bream juveniles with an average body weight of 5 g. The method of immunization was by short bath and three different bacterins against the marine pathogen Photobacterium damselae subsp. piscicida were designed and used to immunize fish. Five genes encoding for five different molecules were analyzed by real time PCR: IL-1ß, IL Ir-2, Cox-2, Mx and TNFα. Gene expression was quantified along four days after fish immunization and results were compared among groups. Results show that the heat-inactivated vaccine stimulates the up-regulation of IL-1ß, IL Ir-2, Cox-2 and TNFα genes whereas the UV-light inactivated vaccine was the unique vaccine which stimulates the expression of Mx gene. The present is a novel study that shows by the first time the effect of the inactivation process of vaccines on the expression levels of genes involved in the defense against Photobacterium damselae subsp piscicida.

Poly(ε-caprolactone) films with favourable properties for neural cell growth.

The regeneration of brain tissue is one of the major challenges in regenerative medicine due to the lack of viable grafts to support the re-growth of functional tissue after a traumatic injury. The development of biocompatible and biodegradable structures with appropriate morphology for the interaction with neural tissue is required. The objective pursued in this work is to develop a biodegradable 2D scaffold structure for neural tissue engineering. Poly(ε-caprolactone) (PCL) was the selected material due to its biocompatibility and biodegradability in the long term. PCL (15%w/w) was dissolved in N-methylpyrrolidone and the film was fabricated by phase inversion casting technique employing ethanol and isopropanol as coagulation baths. The physical structure, morphology and topography of the flat scaffolds were characterized using different techniques. The two different scaffolds presented homogeneous structure with high porosity (higher than 85%), contact angles higher than 90(o), high roughness (Ra> 0.6 µm) and superficial pore sizes of 0.7 and 1.7 µm, respectively. Permeance tests showed high water permeabilities (~350-590 mL m(-1) bar(-1) h(-1)) indicative of promising nutrients supply to the cells. Finally, in vitro human glioblastoma cells cultures after 48 hours showed good cell attachment, proliferation and penetration in the scaffolds. Detailed evaluation of the interaction between the surface morphology and the properties of the scaffolds with the cell response has been done. Thus, the PCL films herein fabricated show promising results as scaffolds for neural tissue regeneration.

Interaction of Corynebacterium pseudotuberculosis with ovine cells in vitro.

Caseous lymphadenitis is an infectious and contagious disease caused by Corynebacterium pseudotuberculosis, with a worldwide distribution and high prevalence in small ruminant populations. This disease causes significant economic loss in small ruminants through reduced meat, wool, and milk production. C. pseudotuberculosis can also affect horses, domestic and wild large ruminants, swine, and man. It is considered an occupational zoonosis for humans. As part of in vitro investigations of the pathogenesis of C. pseudotuberculosis, this study analyzed its capacity to adhere to and invade the FLK-BLV-044 cell line, derived from ovine embryonic kidney cells. C. pseudotuberculosis showed a measurable capacity to adhere to and invade this cell line with no significant differences between the four strains assessed. The incubation of the cell line at 4ºC, pre-incubation with sugars, complete and heat inactivated antiserum, and heat-killed and ultraviolet-killed bacteria produced a significant (P < 0.05) decrease in the invasion efficiency or inability to invade the cell line. Plate counting and fluorescence studies showed intracellular bacteria for up to 6 days. Non-phagocytic cells may therefore act as a suitable environment for C. pseudotuberculosis survival and play a role in the spread of infection and/or maintenance of a carrier state.

In vitro study of adherence, invasion, and persistence of Streptococcus iniae in fibroblastic-like fish cell line SAF-1.

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Gentamicin protection assays were used to investigate the ability of different S. iniae strains to invade and adhere to fibroblastic-like fish cell line SAF-1. All strains tested were detected intracellularly using both techniques, with variable internalization degrees between strains. The experiments carried out at 4°C demonstrated that active cell metabolism is necessary for bacterial internalization. Intracellular bacteria were detected for up to 3 d with a round morphology and were stained with 4',6-diamidino-2-phenylindole (DAPI), indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that S. iniae is capable of adhering, entering, and surviving within fibroblastic cells, which may be important for the persistence and establishment of a carrier state.

Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells.

Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues.