- Email: [email protected]
Acinetobacter pittii biofilm formation on inanimate surfaces after long-term desiccation
Accepted Manuscript Acinetobacter pittii maintains its ability to form biofilms on inanimate surfaces after long-term desiccation Zaloa Bravo, Itziar Chapartegui-González, María Lázaro-Díez, José Ramos-Vivas PII:
S0195-6701(17)30413-9
DOI:
10.1016/j.jhin.2017.07.031
Reference:
YJHIN 5186
To appear in:
Journal of Hospital Infection
Received Date: 7 April 2017 Revised Date:
0195-6701 0195-6701
Accepted Date: 25 July 2017
Please cite this article as: Bravo Z, Chapartegui-González I, Lázaro-Díez M, Ramos-Vivas J, Acinetobacter pittii maintains its ability to form biofilms on inanimate surfaces after long-term desiccation, Journal of Hospital Infection (2017), doi: 10.1016/j.jhin.2017.07.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Acinetobacter pittii maintains its ability to form biofilms on inanimate surfaces after
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long-term desiccation
3 Running title: Acinetobacter pittii survival on surfaces
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Zaloa Bravo 1,2,γ, Itziar Chapartegui-González1,3, γ, María Lázaro-Díez1,3,4, and José
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Ramos-Vivas1,3,4* 1
Instituto de Investigación Valdecilla IDIVAL, Santander, 39011, Spain
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Department of Immunology, Microbiology and Parasitology. Faculty of Science and
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Technology. University of the Basque Country. Leioa, 48940, Spain
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39008, Spain
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III, Madrid, 28029, Spain
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γ
Z.B.-H and I.C.G. contributed equally to this work.
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Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos
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Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Santander,
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* Corresponding author. Tel.: +34 942 315515-Ext 73172; fax: +34 942 315517.
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E-mail address: [email protected]
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ACCEPTED MANUSCRIPT Summary
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Background
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The survival of pathogenic microorganisms in the health care environment has a major
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role in nosocomial infections. Among the responsible mechanisms that could allow
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nosocomial pathogens to persist with these stress conditions are their ability to resist
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desiccation and to form biofilms.
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Aim. To investigate the survival behaviour of Acinetobacter pittii isolates on inert
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surfaces and saline microcosms.
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Methods. Five A. pittii clinical strains were spotted over white lab coat fragments,
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glass and plastic surfaces, or inoculated into sterile saline and monitored at room
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temperature over a period of 43 days.
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Findings. Our results showed that, although the permanence on solid surfaces
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negatively affects the culturability of the studied strains, a fraction of stressed cells
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survived for at least the period of study. On average, A. pittii culturability was reduced
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by 77.3%, 80.9%, and 68.1% in white coat, plastic and glass surfaces, respectively.
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However, approximately 85.6% of the populations retain their culturability in saline
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solution. Culturability correlated with the presence of cells with an intact membrane as
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demonstrated after LIVE/DEAD staining. Supplementation of the culture medium with
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sodium pyruvate favoured the culturability of strains from all conditions but in general,
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A. pittii populations did not enter a viable but non-culturable state.
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Conclusion. After long-term desiccation, all A. pittii strains retained, or even increased,
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their capability to form biofilms once they are feed with nutrient media. This suggests
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that A. pittii is able of an easy recovery from desiccation and could also express
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adherence factors to infect new hosts after rehydration.
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Keywords: Acinetobacter pittii, survival time, biofilm, healthcare-associated infections
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The genus Acinetobacter currently comprises 49 validly named species. Some species
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are closely related, including the members of the so-called Acinetobacter
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calcoaceticus-Acinetobacter baumannii (Acb) complex. Bacteria within the Acb
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complex cause various types of human infections, including pneumonia, bacteraemia,
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wound infections, meningitis and urinary tract infections [1]. The treatment options
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have become extremely limited due to propensity of these species to develop
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resistance to conventional antimicrobial agents. A. baumannii is the most frequently
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occurring Acinetobacter spp. among clinical Acinetobacter isolates, and most hospital
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outbreaks are attributed to this species. Less commonly, A. baumannii can also cause
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community-acquired infections, including pneumonia and bacteraemia [2].
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Although A. pittii is considered less problematic than A. baumannii, this species is
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increasingly reported in clinical specimens [1, 3]. Moreover, the emergence of
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carbapenem-resistant A. pittii strains possessing carbapenem-hydrolyzing β-
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lactamases, such as NDM1, has become a great medical concern [4, 5]. Knowledge of
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Acinetobacter virulence factors or their role in pathogenesis is limited. Also, to the best
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of our knowledge, there are no detailed published reports on virulence factors in A.
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pittii [6, 7]. Furthermore, Acinetobacter spp. are able to rapidly spread among patients
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in the hospital settings. One important factor contributing to the spread of
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Acinetobacter in these environments is its propensity to withstand desiccation and to
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survive on inanimate surfaces [8, 9]. Inert surfaces in hospitals (e.g., walls, furniture
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and equipment/medical devices) have often been described as the source for
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outbreaks of nosocomial infections and contaminated objects include hospital bed rails
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and bed linen, curtains, mattresses, patients' gowns and clothing, tables, towels,
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etc.[10]. In this way, a single hand contact with a contaminated surface results in a
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variable degree of pathogen transfer [11].
78 A better understanding of Acinetobacter spp. survival in the hospital environment
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could provide key information to understand their epidemiology and to control their
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spread. It is thus of interest to investigate the responses of Acinetobacter spp. to
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conditions similar to those found in the hospital environment. Bacteria can attach to
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inert surfaces and survive desiccation, thus we believe that it is important to know
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what happens when stressed bacteria colonize or infect a new host again resulting in a
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shift towards a nutrient-rich and warm environment. There is a gap in the knowledge
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on the bacterial transition from a stressed state (i.e. ex-vivo, on inanimate surfaces) to
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a new environment with available nutrients and higher temperature (inside a new
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host). Those bacteria under stress should conserve factors that favour infection. In the
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present study, the behaviour of cells of A. pittii clinical isolates under starvation and
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desiccation conditions was evaluated. Furthermore, the maintenance of biofilm
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forming capability of bacteria was examined after addition of nutrients to bacteria
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under desiccation stress.
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Bacterial strains and inoculum preparation
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Five A. pittii clinical isolates used in this work are listed in Table I, and were all
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previously described [7]. Reference strain A. pittii LMG 10559 was also included. The
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strains were routinely cultured on Luria Bertani (LB) broth or agar at 37°C, and frozen
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at -80°C with 20% glycerol. Strains were grown at 37°C with shaking (200 rpm) for 24 h.
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Cells were collected by centrifugation (2500 g for 20 min) and washed three times with
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sterile saline solution (0.9 % wt/vol NaCl). Finally, cell pellets were suspended in sterile
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saline solution.
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104 Survival assays
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A. pittii cells from stationary phase were incubated at 21.8±0.23ºC in liquid or solid
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environments. Starvation was implemented by incubating cells in sterile saline solution
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or onto cotton white lab coat fragments (~1 cm2), plastic (bottom of wells in 24-well
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plates (1.9 cm2, CorningTM CostarTM, Fisher Scientific), or sterile glass cover slides (12
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mm diameter, 1.13 cm2). For survival assays in the aqueous environment, experiments
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were carried out in 50 ml polypropylene tubes (Fisher Scientific) containing 15 ml of
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sterile saline solution reaching a bacterial density of ~107 cells/ml.
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For survival assays onto solid surfaces, glass cover slides, plastic or white lab coat
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fragments were inoculated with 50 µl spots of A. pittii at the same cell density. Cover
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slides and lab coat fragments were sterilized by autoclaving (121ºC/20 min) and 24
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well plates were purchased from Fisher Scientific (individually wrapped, sterilized by
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gamma irradiation). 6
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Cover slides and fragments of lab coats were placed in wells of several 24-well plates in
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the
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hygrometer/thermometer (Thermo Hygro) and maintained at a relatively low level (54
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± 1.6% humidity) and room temperature 21.8 ± 0.23ºC. To recover populations from
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solid surfaces, 1 ml of saline solution was added to glass or plastic surfaces and the
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cells were scraped off from the bottom using a 1 ml tip. To recover A. pittii from the
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white coat, the lab coat fragments were placed in a polypropylene tube with 5 ml
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saline solution and vigorously shaken with a vortex. During the experiments, samples
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from solid surfaces and saline microcosms were collected at different time points (days
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0, 1, 2, 3, 8, 15, 22, 29, 36, 43), serially diluted and used to inoculate LB agar or LB agar
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amended with sodium pyruvate (1% wt/vol) [12, 13] to determine the number of
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colony-forming units. Sodium pyruvate was added directly to LB agar prior to
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autoclaving.
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Three independent experiments were performed. Along desiccation survival period
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and to determine the regrowth capacity, recovered A. pittii cells were resuspended in
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LB broth and incubated for 48 h at 37ºC. At the end of the new incubation period,
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optical density (OD) at 620 nm was recorded. Purity of cultures was confirmed by
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visual
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immunofluorescence staining with a specific antiserum against A. pittii [14].
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inspection
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morphology
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plating
and
by
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Biofilm formation
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Biofilm formation was estimated after addition of 1 ml of LB medium to all solid
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surfaces contained on 24-well plates according to previously described protocols [14]. 7
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37°C without shaking. Planktonic cells were removed, and wells were rinsed three
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times with distilled water, and the remaining adherent bacteria were stained with 1.5
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ml/well of crystal violet (CV) (0.7% [wt/vol] solution) for 12 min. Excess stain was
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removed and CV was extracted with 1.5 ml acetic acid (33%). Plates were incubated at
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room temperature in an orbital shaker for 1 min at 400 rpm to release the dye into the
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solution. Then a sample of 100 µl was transferred to another 96-well flat-bottom plate,
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and the amount of dye (proportional to the density of adherent cells) was determined
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at 620 nm using a microplate reader (Multiskan FC; Thermo Fisher). In each
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experiment, results were corrected for background staining by subtracting the value
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for crystal violet bound to uninoculated controls. The biofilm assay was performed
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three times, with duplicates in each assay. To quantify specific biofilm-forming ability,
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the number of planktonic cells (CFUs) was converted to a logarithmic scale, and
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normalized biofilms were calculated by dividing the total biofilm value (expressed as
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the OD620) by the bacterial growth for each strain (expressed in CFUs).
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Confocal Laser Scanning Microscopy (CLSM)
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Bacteria viability during the survival assays on cover slides was determined as
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previously described [14]. Briefly, bacterial viability was determined by using the
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BacLight LIVE/DEAD kit (Molecular Probes Inc.). Bacteria were stained for 20 min at
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room temperature in the dark. A series of optical sections was obtained with a Nikon
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A1R confocal scanning laser microscope (CLSM); the excitation wavelengths were 488
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nm (green) and 561 nm (red), and 500- to 550-nm and 570- to 620-nm emission filters
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were used, respectively. Images were captured at random with a 20× Plan Apo
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assembled using NIS-Elements software, version 3.2. We have used the volume
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measurement tool of the Nis-elements software to easily recognize the relative
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biomass of live (green fluorescence) and dead cells (red fluorescence) expressed as
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µm3. We have calculated the percentage of biomass that was alive, and the percentage
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of biomass that was dead in all z-stack images from representative assays.
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173 Statistics
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All data were derived from three independent experiments. Statistical analysis of the
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data was carried out with the paired two-tailed Student t-test. A p-value less than 0.05
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was considered statistically significant.
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Figure 1 shows the culturability of the A. pittii strains in solid surfaces and saline
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solution over a 43 days period. On average, A. pittii culturability was reduced by 77.3%,
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80.9%, 68.1% and 14.5% in white coat, plastic, glass and saline respectively. Significant
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statistical differences (p < 0.05) were found between strain LMG 10559 and HUMV
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5918 in plastic and lab coat; between strain LMG 10559 and strain HUMV 6483 in lab
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coat; and between strain HUMV 6207 and strains HUMV 5918 and HUMV 6483 in the
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lab coat. No statistical significant differences were shown between strains in saline or
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glass.
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In two strains (5918 and 6483, isolated from a wound exudate and urine respectively)
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the culturability was only reduced in 53.5% and 54.1% in the white coat, indicating that
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nearly half the population survive on this surface for at least 6 weeks. Both strains also
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showed greater survival in glass, plastic and saline environment. On the contrary,
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reference strain A. pittii LMG 10559 showed the lowest survival capacity under
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desiccation conditions. In experiments carried out in plastic and white coat solid
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surfaces, colony-forming units declined below the detection limit.
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When colony-forming units were enumerated on LB agar supplemented with sodium
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pyruvate (recovery medium), only a slight positive effect on culturability was observed
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in all strains but it was not statistically significant, p>0.05 (Figure 2). On average, A.
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pittii culturability in this recovery medium was reduced by 74.3%, 79.7%, 70.3% and
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14% in white coat, plastic, glass and saline respectively. Again, the two isolates 5918
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and 6483 showed great survival in white coat and plastic; and in addition, strain 6483 10
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in glass and saline. The survival of both strains in white coat was reduced in 48.4% and
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48.6% respectively.
211 Staining of A. pittii strains spotted on glass by using the BacLight LIVE/DEAD bacterial
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viability kit (Figure 3) demonstrated the transition from largely viable populations (0
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days, most of the cells fluoresced in green) to dead populations (red fluorescent cells).
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So, the presence of live bacteria could be detected after 43 days. Moreover,
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population dynamics differed between strains. After 15 days of desiccation, a large
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proportion of the cells of the strain LMG 10559 fluoresced in red while, most of the
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populations of HUMV 5918 and HUMV 6483 remained viable (Figure 3). Data from all
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strains is presented in Supplementary Figure 1.
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We added 1 ml of LB medium inside the wells containing white lab coat fragments,
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glass and plastic (bottom of wells) to study the ability of these remaining live
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populations to resume growth. Despite a great reduction in viability over time,
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populations recovered from starvation on solid surfaces experiments retained their
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ability to regrowth (as measured by DO620) after rehydration with culture medium
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(Supplementary Figure 2). CFUs were also counted at 0 h, 12 h, 24 h and 48 h post-
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hydration for each strain in each condition. Representative experiments for 3 strains
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are shown in Figure 4. We next tested the capability of these new populations to form
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biofilms by crystal violet assays (Figure 5). The number of CFUs was converted to a
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logarithmic scale, and normalized biofilms were calculated by dividing the total biofilm
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value (expressed as the OD620) by the bacterial growth for each strain (expressed in
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CFUs) (Figure 5). Our results indicate that these populations retain, or even increase,
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their ability to form biofilms after rehydration. Importantly, in both experiments, LMG
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10559 regrew and formed biofilms even when culturable cells were below the
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detection limit (T = 43 days).
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Although the interest in Acinetobacter spp. as nosocomial pathogens has increased,
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little is known about their natural reservoirs and mode of transmission. Moreover, the
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ability of Acinetobacter spp. to survive on dry surfaces is central to our understanding
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of their epidemiology. One important factor contributing to the spread of
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Acinetobacter in hospital settings is its propensity to withstand desiccation and to
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survive on inert surfaces [8, 9]. Wendt et al., have shown that the ability of A.
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baumannii strains to survive under dry conditions which correlated with the source of
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the strains. In that study, strains isolated from dry sources (pillow and a working
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surface) had better survival rates than strains isolated from wet sources (sewage and
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urine) [15]. According to several other authors, the ability of different strains of A.
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baumannii to survive desiccation appears to vary greatly; some strains were able to
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survive for several months without any reduction in colony counts, whereas other
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strains hardly survived the process of drying [11, 15-18].
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In contrast to A. baumannii, there are no detailed published reports on persistence of
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A. pittii clinical strains in the hospital settings. To learn more about its survival strategy
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and capacity to persist in that environment, we studied the effects of nutrient
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deprivation and dryness on the long-term survival of several A. pittii human clinical
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isolates and of a reference strain. Taking into account the fact that different surfaces
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may influence the survival times, we used materials that are typical for the hospital
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environment, including a white lab coat used by professionals in the medical field and
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those involved in laboratory work. Our experiments with A. pittii cells retained on dry
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surfaces revealed the long-term survival was not influenced by the material because a
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constant reduction in culturability was observed on cotton, plastic and glass surfaces.
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The resistance to stressful conditions was moderate; with survival on dry surfaces
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being strain-dependent; and the reference strain A. pittii LMG, the most sensitive to
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desiccation.
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Jawad et al., studied the influence of relative humidity on survival of two A. pittii
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strains on dry surfaces [8]. They used one strain isolated from human cerebrospinal
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fluid and another from an unspecified source. These strains survived at room
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temperature for up to 5 or 10 days respectively when bacteria were suspended in
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distilled water and placed in glass cover slips. Staining of A. pittii strains spotted on
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glass clearly demonstrated the presence of cells with intact cell membranes after long
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term survival. As a decline in culturability after long periods of incubation in several
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microcosms may be due either to cell death or to entry into a viable but non-culturable
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(VBNC) state, we tested a method for in vitro resuscitation. As sodium pyruvate and
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H2O2-degrading agents seem to be effective compounds for recovering the
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physiological state after cell damage or a loss of culturability [17, 19], we wanted to
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know if there is any effect on culturability in A. pittii stressed cells by adding sodium
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pyruvate to the recovery medium. The loss of viability and culturability in this solid
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surface indicates that a great fraction of population underwent cellular membrane
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damaged and the VBNC state was not induced under these conditions.
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We clearly found that A. pittii survival in the liquid environment was greater than in
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solid surfaces. On average, A. pittii culturability was reduced by more than 68% in solid
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surfaces but only by 14.5% in saline. Our results with A. pittii on dry surfaces are in 14
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culturability of A. baumannii population did not experience significant changes for at
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least one month [9, 15, 17]. Otherwise, any conclusions on the correlation between
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survival under laboratory conditions resembling hospital settings must take into
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account the fact that most transmission occurs via the hands of healthcare workers.
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These hands transport bacteria from inert surfaces to patients [20]. Once transported,
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stressed bacteria must be able to recover from their state by a fast adaptation to a
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new warm and nutrient-rich environment inside a new patient. As those bacteria
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under stress should conserve factors that favour infection, we wanted to know
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bacterial behaviour after rehydration with rich media containing nutrients, in a warm
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temperature (37ºC), simulating conditions close to what could happen upon entering a
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new patient/host.
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Interestingly, despite a great reduction in culturability over time, all strains are able to
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maintain their capacity to form biofilms after rehydration, addition of nutrients and
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changing temperature. Rehydration enables cells to recover membrane fluidity
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associated with cell proliferation capabilities [21]. The bacteria demonstrated a rapid
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adaptation to the temperature shift (from room temperature to 37ºC) and nutrient
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availability (from starvation to food availability); conditions that may easily be found in
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a new patient host. The data presented contribute to a better understanding of the
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behaviour of A. pittii on inanimate surfaces. Our findings are also consistent with the
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observed tendency of other Acinetobacter species to survive on dry surfaces, and their
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ability to be transferred under dry conditions in a hospital environment during
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nosocomial infection outbreaks.
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Survival of the two A. pittii strains on white lab coat fragments is remarkable. In a
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recent study, Munoz-Price found concordance between contamination of hands of
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health care workers and their white coats [22]. Their results include strains of
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Acinetobacter spp. as found as contaminants.
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As patients continuously shed infectious bacteria in the hospital environment and
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health professionals are in constant contact with these patients, there is a need to
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promote scrupulous hand washing and to search for alternatives to white coats [23]. In
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addition, health care workers often walk from healthcare facilities to other public
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places such as cafeterias and hospital annexes wearing their work clothing, creating
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another route by which micro-organisms can be exported from and imported into the
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healthcare environment [23, 24]. Although there are no studies that directly link white
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coats with infection transmission in Acinetobacter, the fact that they could be
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contaminated, and transport viable and culturable pathogens over time suggests the
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need for further research.
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Acknowledgements
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Z.B.-H. holds a contract from the University of the Basque Country. I.C.-G. holds a
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contract
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(AIP14/01/PI13/01310). M.L.-D. holds a contract from the Instituto de Investigación
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Sanitaria Valdecilla IDIVAL and Universidad de Cantabria (PREVAL16/05). The authors
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thank Dr. Fidel Madrazo (Technology Support Services, IDIVAL) for helping with
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confocal microscopy and Dr Inés Arana for his excellent assistance in the preparation
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of the manuscript. J. R.-V. acknowledges the receipt of a SEIMC fellowship. Research in
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our laboratory is supported by the Spanish Instituto de Salud Carlos III, Spain (grants
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PI13/01310 and PI16/01103) and the Plan Nacional de I+D+i 2008-2011 and Instituto
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de Salud Carlos III, Subdirección General de Redes y Centros de Investigación
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Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research
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in Infectious Diseases (REIPI RD12/0015) - co-financed by European Development
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Regional Fund "A way to achieve Europe" ERDF.
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Acinetobacter baumannii on dry surfaces. The Journal of hospital infection
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Oliver JD Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS microbiology reviews 2010; 34: 415-25.
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Kramer A, Schwebke I, Kampf G How long do nosocomial pathogens persist on
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2014; 5: 258.
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Remuzgo-Martinez S, Lazaro-Diez M, Mayer C et al. Biofilm Formation and
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Quorum-Sensing-Molecule
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liquefaciens. Applied and environmental microbiology 2015; 81: 3306-15.
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Wendt C, Dietze B, Dietz E, Ruden H Survival of Acinetobacter baumannii on dry surfaces. Journal of clinical microbiology 1997; 35: 1394-7.
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Jawad A, Heritage J, Snelling AM, Gascoyne-Binzi DM, Hawkey PM Influence of
relative humidity and suspending menstrua on survival of Acinetobacter spp. on dry surfaces. Journal of clinical microbiology 1996; 34: 2881-7.
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Bravo Z, Orruno M, Parada C, Kaberdin VR, Barcina I, Arana I The long-term survival of Acinetobacter baumannii ATCC 19606(T) under nutrient-deprived
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conditions does not require the entry into the viable but non-culturable state.
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Archives of microbiology 2016; 198: 399-407.
396
[18]
Gastmeier P, Schwab F, Barwolff S, Ruden H, Grundmann H Correlation between the genetic diversity of nosocomial pathogens and their survival time
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in intensive care units. The Journal of hospital infection 2006; 62: 181-6.
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Vivas J, Carracedo B, Riano J et al. Behavior of an Aeromonas hydrophila aroA live vaccine in water microcosms. Applied and environmental microbiology
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intensive care units. The American journal of medicine 1991; 91: 179S-84S.
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[21]
Lemke MJ, Leff LG Culturability of stream bacteria assessed at the assemblage and population levels. Microbial ecology 2006; 51: 365-74.
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Weinstein RA Epidemiology and control of nosocomial infections in adult
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[22]
Munoz-Price LS, Arheart KL, Mills JP et al. Associations between bacterial
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contamination of health care workers' hands and contamination of white coats
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and scrubs. American journal of infection control 2012; 40: e245-8. [23]
healthcare textiles in the transmission of pathogens: a review of the literature.
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Mitchell A, Spencer M, Edmiston C, Jr. Role of healthcare apparel and other
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The Journal of hospital infection 2015; 90: 285-92.
[24]
Wong D, Nye K, Hollis P Microbial flora on doctors' white coats. BMJ (Clinical
research ed 1991; 303: 1602-4.
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Table 1. Acinetobacter pittii strains used in this study.
419 Clinical source
1
A. pittii
a
LMG 10559
tracheal aspirate
2
A. pittii
b
HUMV 0315
sputum
3
A. pittii
HUMV 4336
diabetic foot exudate
4
A. pittii
HUMV 6207
wound exudate
5
A. pittii
HUMV 5918
wound exudate
6
A. pittii
HUMV 6483
urine
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Species
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420 421
a
LMG, Culture Collection at Laboratorium voor Microbiologie Gent.
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b
HUMV, Culture Collection at Hospital Universitario Marqués de Valdecilla.
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Figure 1.
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Survival times of strains inoculated onto white lab coat, plastic or in glass coverslips
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(54.3% relative humidity) or suspended in sterile saline solution, and kept at room
439
temperature. Colony counts on Luria Bertani agar determined cell survival. Each point
440
represents the mean of three independent experiments expressed as percentage of
441
reduction in culturability with respect to day 0 (100%).
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Figure 2.
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Survival times of strains inoculated onto white lab coat, plastic or in glass coverslips
445
(54.3% relative humidity) or suspended in saline and kept at room temperature.
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Colony counts on Luria Bertani agar supplemented with sodium pyruvate determined
447
cell survival. Each point represents the mean of three independent experiments
448
expressed as percentage of reduction in culturability with respect to day 0 (100%).
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Figure 3.
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CLSM images of strains LMG, 5918 and 6483 after survival onto glass cover slips during
452
different times. Bacteria were stained with the LIVE/DEAD viability kit. Live cells
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fluoresce in green with Syto 9 dye and dead cells are stained red with propidium
454
iodide. Arrows indicate a magnification (right panel) from a representative area of 43
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days. Original magnification: ×600. Lower panel: fluorescence (live/dead) for each
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strain represented in the upper panel, expressed as percentage.
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Figure 4.
460
Estimation of regrowth capacity by enumeration of colony-forming units in three
461
strains. The CFUs were counted at 0 h, 12 h, 24 h and 48 h post-hydration for each
462
strain, in each condition.
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465
Biofilm formation by A. pittii strains after desiccation and rehydration with Luria
466
Bertani (LB) medium. Shown are representative examples of biofilm formation in 24-
467
well plates by the 6 A. pittii strains spotted onto various surfaces after rehydration and
468
growth in LB medium for 48h at 37°C. Wells were stained with crystal violet (upper
469
panel). Quantification of biofilm formation was performed after crystal violet
470
extraction and measurement (OD620). Lower panel: normalized biofilm formation,
471
calculated as total biofilm (expressed as the OD620) divided by growth (expressed in
472
CFUs). Values are presented as mean ± standard deviation (SD) of three independent
473
experiments. Asterisks indicate: *, p<0.05; **, p<0.01.
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Supplementary Figure 1.
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Confocal scanning laser microscopy. Fluorescence (live/dead) expressed as percentage,
477
for each strain, at different time points.
478
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Supplementary Figure 2. Regrowth capacity estimated for A pittii populations
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maintained on solid surfaces at time 0 or at different time points after desiccation.
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Stressed populations were resuspended in LB medium, incubated for 48h at 37ºC and
482
the OD620 of bacterial cultures was measured. Columns, from white to black, strains 23
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LMG, HUMV 0315, HUMV 4336, HUMV 6207, HUMV 5918 and HUMV 6483. Values are
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presented as mean ± standard deviation (SD) of three independent experiments.
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S0195-6701(17)30413-9
DOI:
10.1016/j.jhin.2017.07.031
Reference:
YJHIN 5186
To appear in:
Journal of Hospital Infection
Received Date: 7 April 2017 Revised Date:
0195-6701 0195-6701
Accepted Date: 25 July 2017
Please cite this article as: Bravo Z, Chapartegui-González I, Lázaro-Díez M, Ramos-Vivas J, Acinetobacter pittii maintains its ability to form biofilms on inanimate surfaces after long-term desiccation, Journal of Hospital Infection (2017), doi: 10.1016/j.jhin.2017.07.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Acinetobacter pittii maintains its ability to form biofilms on inanimate surfaces after
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long-term desiccation
3 Running title: Acinetobacter pittii survival on surfaces
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Zaloa Bravo 1,2,γ, Itziar Chapartegui-González1,3, γ, María Lázaro-Díez1,3,4, and José
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Ramos-Vivas1,3,4* 1
Instituto de Investigación Valdecilla IDIVAL, Santander, 39011, Spain
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2
Department of Immunology, Microbiology and Parasitology. Faculty of Science and
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Technology. University of the Basque Country. Leioa, 48940, Spain
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3
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39008, Spain
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4
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III, Madrid, 28029, Spain
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γ
Z.B.-H and I.C.G. contributed equally to this work.
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Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos
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Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Santander,
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* Corresponding author. Tel.: +34 942 315515-Ext 73172; fax: +34 942 315517.
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E-mail address: [email protected]
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ACCEPTED MANUSCRIPT Summary
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Background
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The survival of pathogenic microorganisms in the health care environment has a major
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role in nosocomial infections. Among the responsible mechanisms that could allow
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nosocomial pathogens to persist with these stress conditions are their ability to resist
30
desiccation and to form biofilms.
31
Aim. To investigate the survival behaviour of Acinetobacter pittii isolates on inert
32
surfaces and saline microcosms.
33
Methods. Five A. pittii clinical strains were spotted over white lab coat fragments,
34
glass and plastic surfaces, or inoculated into sterile saline and monitored at room
35
temperature over a period of 43 days.
36
Findings. Our results showed that, although the permanence on solid surfaces
37
negatively affects the culturability of the studied strains, a fraction of stressed cells
38
survived for at least the period of study. On average, A. pittii culturability was reduced
39
by 77.3%, 80.9%, and 68.1% in white coat, plastic and glass surfaces, respectively.
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However, approximately 85.6% of the populations retain their culturability in saline
41
solution. Culturability correlated with the presence of cells with an intact membrane as
42
demonstrated after LIVE/DEAD staining. Supplementation of the culture medium with
43
sodium pyruvate favoured the culturability of strains from all conditions but in general,
44
A. pittii populations did not enter a viable but non-culturable state.
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Conclusion. After long-term desiccation, all A. pittii strains retained, or even increased,
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their capability to form biofilms once they are feed with nutrient media. This suggests
47
that A. pittii is able of an easy recovery from desiccation and could also express
48
adherence factors to infect new hosts after rehydration.
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Keywords: Acinetobacter pittii, survival time, biofilm, healthcare-associated infections
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The genus Acinetobacter currently comprises 49 validly named species. Some species
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are closely related, including the members of the so-called Acinetobacter
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calcoaceticus-Acinetobacter baumannii (Acb) complex. Bacteria within the Acb
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complex cause various types of human infections, including pneumonia, bacteraemia,
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wound infections, meningitis and urinary tract infections [1]. The treatment options
57
have become extremely limited due to propensity of these species to develop
58
resistance to conventional antimicrobial agents. A. baumannii is the most frequently
59
occurring Acinetobacter spp. among clinical Acinetobacter isolates, and most hospital
60
outbreaks are attributed to this species. Less commonly, A. baumannii can also cause
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community-acquired infections, including pneumonia and bacteraemia [2].
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Although A. pittii is considered less problematic than A. baumannii, this species is
64
increasingly reported in clinical specimens [1, 3]. Moreover, the emergence of
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carbapenem-resistant A. pittii strains possessing carbapenem-hydrolyzing β-
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lactamases, such as NDM1, has become a great medical concern [4, 5]. Knowledge of
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Acinetobacter virulence factors or their role in pathogenesis is limited. Also, to the best
68
of our knowledge, there are no detailed published reports on virulence factors in A.
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pittii [6, 7]. Furthermore, Acinetobacter spp. are able to rapidly spread among patients
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in the hospital settings. One important factor contributing to the spread of
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Acinetobacter in these environments is its propensity to withstand desiccation and to
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survive on inanimate surfaces [8, 9]. Inert surfaces in hospitals (e.g., walls, furniture
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and equipment/medical devices) have often been described as the source for
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outbreaks of nosocomial infections and contaminated objects include hospital bed rails
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and bed linen, curtains, mattresses, patients' gowns and clothing, tables, towels,
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etc.[10]. In this way, a single hand contact with a contaminated surface results in a
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variable degree of pathogen transfer [11].
78 A better understanding of Acinetobacter spp. survival in the hospital environment
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could provide key information to understand their epidemiology and to control their
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spread. It is thus of interest to investigate the responses of Acinetobacter spp. to
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conditions similar to those found in the hospital environment. Bacteria can attach to
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inert surfaces and survive desiccation, thus we believe that it is important to know
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what happens when stressed bacteria colonize or infect a new host again resulting in a
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shift towards a nutrient-rich and warm environment. There is a gap in the knowledge
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on the bacterial transition from a stressed state (i.e. ex-vivo, on inanimate surfaces) to
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a new environment with available nutrients and higher temperature (inside a new
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host). Those bacteria under stress should conserve factors that favour infection. In the
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present study, the behaviour of cells of A. pittii clinical isolates under starvation and
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desiccation conditions was evaluated. Furthermore, the maintenance of biofilm
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forming capability of bacteria was examined after addition of nutrients to bacteria
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under desiccation stress.
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Bacterial strains and inoculum preparation
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Five A. pittii clinical isolates used in this work are listed in Table I, and were all
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previously described [7]. Reference strain A. pittii LMG 10559 was also included. The
99
strains were routinely cultured on Luria Bertani (LB) broth or agar at 37°C, and frozen
100
at -80°C with 20% glycerol. Strains were grown at 37°C with shaking (200 rpm) for 24 h.
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Cells were collected by centrifugation (2500 g for 20 min) and washed three times with
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sterile saline solution (0.9 % wt/vol NaCl). Finally, cell pellets were suspended in sterile
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saline solution.
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104 Survival assays
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A. pittii cells from stationary phase were incubated at 21.8±0.23ºC in liquid or solid
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environments. Starvation was implemented by incubating cells in sterile saline solution
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or onto cotton white lab coat fragments (~1 cm2), plastic (bottom of wells in 24-well
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plates (1.9 cm2, CorningTM CostarTM, Fisher Scientific), or sterile glass cover slides (12
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mm diameter, 1.13 cm2). For survival assays in the aqueous environment, experiments
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were carried out in 50 ml polypropylene tubes (Fisher Scientific) containing 15 ml of
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sterile saline solution reaching a bacterial density of ~107 cells/ml.
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For survival assays onto solid surfaces, glass cover slides, plastic or white lab coat
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fragments were inoculated with 50 µl spots of A. pittii at the same cell density. Cover
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slides and lab coat fragments were sterilized by autoclaving (121ºC/20 min) and 24
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well plates were purchased from Fisher Scientific (individually wrapped, sterilized by
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gamma irradiation). 6
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Cover slides and fragments of lab coats were placed in wells of several 24-well plates in
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the
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hygrometer/thermometer (Thermo Hygro) and maintained at a relatively low level (54
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± 1.6% humidity) and room temperature 21.8 ± 0.23ºC. To recover populations from
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solid surfaces, 1 ml of saline solution was added to glass or plastic surfaces and the
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cells were scraped off from the bottom using a 1 ml tip. To recover A. pittii from the
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white coat, the lab coat fragments were placed in a polypropylene tube with 5 ml
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saline solution and vigorously shaken with a vortex. During the experiments, samples
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from solid surfaces and saline microcosms were collected at different time points (days
128
0, 1, 2, 3, 8, 15, 22, 29, 36, 43), serially diluted and used to inoculate LB agar or LB agar
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amended with sodium pyruvate (1% wt/vol) [12, 13] to determine the number of
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colony-forming units. Sodium pyruvate was added directly to LB agar prior to
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autoclaving.
Ambient
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Three independent experiments were performed. Along desiccation survival period
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and to determine the regrowth capacity, recovered A. pittii cells were resuspended in
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LB broth and incubated for 48 h at 37ºC. At the end of the new incubation period,
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optical density (OD) at 620 nm was recorded. Purity of cultures was confirmed by
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visual
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immunofluorescence staining with a specific antiserum against A. pittii [14].
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inspection
on
their
colonial
morphology
after
plating
and
by
139 140
Biofilm formation
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Biofilm formation was estimated after addition of 1 ml of LB medium to all solid
142
surfaces contained on 24-well plates according to previously described protocols [14]. 7
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37°C without shaking. Planktonic cells were removed, and wells were rinsed three
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times with distilled water, and the remaining adherent bacteria were stained with 1.5
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ml/well of crystal violet (CV) (0.7% [wt/vol] solution) for 12 min. Excess stain was
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removed and CV was extracted with 1.5 ml acetic acid (33%). Plates were incubated at
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room temperature in an orbital shaker for 1 min at 400 rpm to release the dye into the
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solution. Then a sample of 100 µl was transferred to another 96-well flat-bottom plate,
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and the amount of dye (proportional to the density of adherent cells) was determined
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at 620 nm using a microplate reader (Multiskan FC; Thermo Fisher). In each
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experiment, results were corrected for background staining by subtracting the value
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for crystal violet bound to uninoculated controls. The biofilm assay was performed
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three times, with duplicates in each assay. To quantify specific biofilm-forming ability,
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the number of planktonic cells (CFUs) was converted to a logarithmic scale, and
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normalized biofilms were calculated by dividing the total biofilm value (expressed as
157
the OD620) by the bacterial growth for each strain (expressed in CFUs).
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Confocal Laser Scanning Microscopy (CLSM)
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Bacteria viability during the survival assays on cover slides was determined as
161
previously described [14]. Briefly, bacterial viability was determined by using the
162
BacLight LIVE/DEAD kit (Molecular Probes Inc.). Bacteria were stained for 20 min at
163
room temperature in the dark. A series of optical sections was obtained with a Nikon
164
A1R confocal scanning laser microscope (CLSM); the excitation wavelengths were 488
165
nm (green) and 561 nm (red), and 500- to 550-nm and 570- to 620-nm emission filters
166
were used, respectively. Images were captured at random with a 20× Plan Apo
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assembled using NIS-Elements software, version 3.2. We have used the volume
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measurement tool of the Nis-elements software to easily recognize the relative
170
biomass of live (green fluorescence) and dead cells (red fluorescence) expressed as
171
µm3. We have calculated the percentage of biomass that was alive, and the percentage
172
of biomass that was dead in all z-stack images from representative assays.
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173 Statistics
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All data were derived from three independent experiments. Statistical analysis of the
176
data was carried out with the paired two-tailed Student t-test. A p-value less than 0.05
177
was considered statistically significant.
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Figure 1 shows the culturability of the A. pittii strains in solid surfaces and saline
187
solution over a 43 days period. On average, A. pittii culturability was reduced by 77.3%,
188
80.9%, 68.1% and 14.5% in white coat, plastic, glass and saline respectively. Significant
189
statistical differences (p < 0.05) were found between strain LMG 10559 and HUMV
190
5918 in plastic and lab coat; between strain LMG 10559 and strain HUMV 6483 in lab
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coat; and between strain HUMV 6207 and strains HUMV 5918 and HUMV 6483 in the
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lab coat. No statistical significant differences were shown between strains in saline or
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glass.
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In two strains (5918 and 6483, isolated from a wound exudate and urine respectively)
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the culturability was only reduced in 53.5% and 54.1% in the white coat, indicating that
197
nearly half the population survive on this surface for at least 6 weeks. Both strains also
198
showed greater survival in glass, plastic and saline environment. On the contrary,
199
reference strain A. pittii LMG 10559 showed the lowest survival capacity under
200
desiccation conditions. In experiments carried out in plastic and white coat solid
201
surfaces, colony-forming units declined below the detection limit.
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When colony-forming units were enumerated on LB agar supplemented with sodium
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pyruvate (recovery medium), only a slight positive effect on culturability was observed
205
in all strains but it was not statistically significant, p>0.05 (Figure 2). On average, A.
206
pittii culturability in this recovery medium was reduced by 74.3%, 79.7%, 70.3% and
207
14% in white coat, plastic, glass and saline respectively. Again, the two isolates 5918
208
and 6483 showed great survival in white coat and plastic; and in addition, strain 6483 10
ACCEPTED MANUSCRIPT 209
in glass and saline. The survival of both strains in white coat was reduced in 48.4% and
210
48.6% respectively.
211 Staining of A. pittii strains spotted on glass by using the BacLight LIVE/DEAD bacterial
213
viability kit (Figure 3) demonstrated the transition from largely viable populations (0
214
days, most of the cells fluoresced in green) to dead populations (red fluorescent cells).
215
So, the presence of live bacteria could be detected after 43 days. Moreover,
216
population dynamics differed between strains. After 15 days of desiccation, a large
217
proportion of the cells of the strain LMG 10559 fluoresced in red while, most of the
218
populations of HUMV 5918 and HUMV 6483 remained viable (Figure 3). Data from all
219
strains is presented in Supplementary Figure 1.
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We added 1 ml of LB medium inside the wells containing white lab coat fragments,
222
glass and plastic (bottom of wells) to study the ability of these remaining live
223
populations to resume growth. Despite a great reduction in viability over time,
224
populations recovered from starvation on solid surfaces experiments retained their
225
ability to regrowth (as measured by DO620) after rehydration with culture medium
226
(Supplementary Figure 2). CFUs were also counted at 0 h, 12 h, 24 h and 48 h post-
227
hydration for each strain in each condition. Representative experiments for 3 strains
228
are shown in Figure 4. We next tested the capability of these new populations to form
229
biofilms by crystal violet assays (Figure 5). The number of CFUs was converted to a
230
logarithmic scale, and normalized biofilms were calculated by dividing the total biofilm
231
value (expressed as the OD620) by the bacterial growth for each strain (expressed in
232
CFUs) (Figure 5). Our results indicate that these populations retain, or even increase,
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their ability to form biofilms after rehydration. Importantly, in both experiments, LMG
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10559 regrew and formed biofilms even when culturable cells were below the
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detection limit (T = 43 days).
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Although the interest in Acinetobacter spp. as nosocomial pathogens has increased,
241
little is known about their natural reservoirs and mode of transmission. Moreover, the
242
ability of Acinetobacter spp. to survive on dry surfaces is central to our understanding
243
of their epidemiology. One important factor contributing to the spread of
244
Acinetobacter in hospital settings is its propensity to withstand desiccation and to
245
survive on inert surfaces [8, 9]. Wendt et al., have shown that the ability of A.
246
baumannii strains to survive under dry conditions which correlated with the source of
247
the strains. In that study, strains isolated from dry sources (pillow and a working
248
surface) had better survival rates than strains isolated from wet sources (sewage and
249
urine) [15]. According to several other authors, the ability of different strains of A.
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baumannii to survive desiccation appears to vary greatly; some strains were able to
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survive for several months without any reduction in colony counts, whereas other
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strains hardly survived the process of drying [11, 15-18].
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In contrast to A. baumannii, there are no detailed published reports on persistence of
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A. pittii clinical strains in the hospital settings. To learn more about its survival strategy
256
and capacity to persist in that environment, we studied the effects of nutrient
257
deprivation and dryness on the long-term survival of several A. pittii human clinical
258
isolates and of a reference strain. Taking into account the fact that different surfaces
259
may influence the survival times, we used materials that are typical for the hospital
260
environment, including a white lab coat used by professionals in the medical field and
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those involved in laboratory work. Our experiments with A. pittii cells retained on dry
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surfaces revealed the long-term survival was not influenced by the material because a
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constant reduction in culturability was observed on cotton, plastic and glass surfaces.
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The resistance to stressful conditions was moderate; with survival on dry surfaces
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being strain-dependent; and the reference strain A. pittii LMG, the most sensitive to
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desiccation.
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Jawad et al., studied the influence of relative humidity on survival of two A. pittii
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strains on dry surfaces [8]. They used one strain isolated from human cerebrospinal
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fluid and another from an unspecified source. These strains survived at room
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temperature for up to 5 or 10 days respectively when bacteria were suspended in
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distilled water and placed in glass cover slips. Staining of A. pittii strains spotted on
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glass clearly demonstrated the presence of cells with intact cell membranes after long
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term survival. As a decline in culturability after long periods of incubation in several
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microcosms may be due either to cell death or to entry into a viable but non-culturable
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(VBNC) state, we tested a method for in vitro resuscitation. As sodium pyruvate and
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H2O2-degrading agents seem to be effective compounds for recovering the
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physiological state after cell damage or a loss of culturability [17, 19], we wanted to
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know if there is any effect on culturability in A. pittii stressed cells by adding sodium
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pyruvate to the recovery medium. The loss of viability and culturability in this solid
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surface indicates that a great fraction of population underwent cellular membrane
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damaged and the VBNC state was not induced under these conditions.
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We clearly found that A. pittii survival in the liquid environment was greater than in
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solid surfaces. On average, A. pittii culturability was reduced by more than 68% in solid
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surfaces but only by 14.5% in saline. Our results with A. pittii on dry surfaces are in 14
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culturability of A. baumannii population did not experience significant changes for at
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least one month [9, 15, 17]. Otherwise, any conclusions on the correlation between
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survival under laboratory conditions resembling hospital settings must take into
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account the fact that most transmission occurs via the hands of healthcare workers.
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These hands transport bacteria from inert surfaces to patients [20]. Once transported,
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stressed bacteria must be able to recover from their state by a fast adaptation to a
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new warm and nutrient-rich environment inside a new patient. As those bacteria
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under stress should conserve factors that favour infection, we wanted to know
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bacterial behaviour after rehydration with rich media containing nutrients, in a warm
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temperature (37ºC), simulating conditions close to what could happen upon entering a
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new patient/host.
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Interestingly, despite a great reduction in culturability over time, all strains are able to
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maintain their capacity to form biofilms after rehydration, addition of nutrients and
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changing temperature. Rehydration enables cells to recover membrane fluidity
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associated with cell proliferation capabilities [21]. The bacteria demonstrated a rapid
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adaptation to the temperature shift (from room temperature to 37ºC) and nutrient
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availability (from starvation to food availability); conditions that may easily be found in
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a new patient host. The data presented contribute to a better understanding of the
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behaviour of A. pittii on inanimate surfaces. Our findings are also consistent with the
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observed tendency of other Acinetobacter species to survive on dry surfaces, and their
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ability to be transferred under dry conditions in a hospital environment during
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nosocomial infection outbreaks.
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Survival of the two A. pittii strains on white lab coat fragments is remarkable. In a
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recent study, Munoz-Price found concordance between contamination of hands of
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health care workers and their white coats [22]. Their results include strains of
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Acinetobacter spp. as found as contaminants.
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As patients continuously shed infectious bacteria in the hospital environment and
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health professionals are in constant contact with these patients, there is a need to
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promote scrupulous hand washing and to search for alternatives to white coats [23]. In
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addition, health care workers often walk from healthcare facilities to other public
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places such as cafeterias and hospital annexes wearing their work clothing, creating
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another route by which micro-organisms can be exported from and imported into the
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healthcare environment [23, 24]. Although there are no studies that directly link white
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coats with infection transmission in Acinetobacter, the fact that they could be
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contaminated, and transport viable and culturable pathogens over time suggests the
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need for further research.
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Acknowledgements
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Z.B.-H. holds a contract from the University of the Basque Country. I.C.-G. holds a
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contract
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(AIP14/01/PI13/01310). M.L.-D. holds a contract from the Instituto de Investigación
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Sanitaria Valdecilla IDIVAL and Universidad de Cantabria (PREVAL16/05). The authors
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thank Dr. Fidel Madrazo (Technology Support Services, IDIVAL) for helping with
334
confocal microscopy and Dr Inés Arana for his excellent assistance in the preparation
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of the manuscript. J. R.-V. acknowledges the receipt of a SEIMC fellowship. Research in
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our laboratory is supported by the Spanish Instituto de Salud Carlos III, Spain (grants
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PI13/01310 and PI16/01103) and the Plan Nacional de I+D+i 2008-2011 and Instituto
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de Salud Carlos III, Subdirección General de Redes y Centros de Investigación
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Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research
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in Infectious Diseases (REIPI RD12/0015) - co-financed by European Development
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Regional Fund "A way to achieve Europe" ERDF.
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the
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Munoz-Price LS, Arheart KL, Mills JP et al. Associations between bacterial
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Table 1. Acinetobacter pittii strains used in this study.
419 Clinical source
1
A. pittii
a
LMG 10559
tracheal aspirate
2
A. pittii
b
HUMV 0315
sputum
3
A. pittii
HUMV 4336
diabetic foot exudate
4
A. pittii
HUMV 6207
wound exudate
5
A. pittii
HUMV 5918
wound exudate
6
A. pittii
HUMV 6483
urine
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420 421
a
LMG, Culture Collection at Laboratorium voor Microbiologie Gent.
422
b
HUMV, Culture Collection at Hospital Universitario Marqués de Valdecilla.
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Figure 1.
437
Survival times of strains inoculated onto white lab coat, plastic or in glass coverslips
438
(54.3% relative humidity) or suspended in sterile saline solution, and kept at room
439
temperature. Colony counts on Luria Bertani agar determined cell survival. Each point
440
represents the mean of three independent experiments expressed as percentage of
441
reduction in culturability with respect to day 0 (100%).
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Figure 2.
444
Survival times of strains inoculated onto white lab coat, plastic or in glass coverslips
445
(54.3% relative humidity) or suspended in saline and kept at room temperature.
446
Colony counts on Luria Bertani agar supplemented with sodium pyruvate determined
447
cell survival. Each point represents the mean of three independent experiments
448
expressed as percentage of reduction in culturability with respect to day 0 (100%).
449
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Figure 3.
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CLSM images of strains LMG, 5918 and 6483 after survival onto glass cover slips during
452
different times. Bacteria were stained with the LIVE/DEAD viability kit. Live cells
453
fluoresce in green with Syto 9 dye and dead cells are stained red with propidium
454
iodide. Arrows indicate a magnification (right panel) from a representative area of 43
455
days. Original magnification: ×600. Lower panel: fluorescence (live/dead) for each
456
strain represented in the upper panel, expressed as percentage.
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Figure 4.
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Estimation of regrowth capacity by enumeration of colony-forming units in three
461
strains. The CFUs were counted at 0 h, 12 h, 24 h and 48 h post-hydration for each
462
strain, in each condition.
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Biofilm formation by A. pittii strains after desiccation and rehydration with Luria
466
Bertani (LB) medium. Shown are representative examples of biofilm formation in 24-
467
well plates by the 6 A. pittii strains spotted onto various surfaces after rehydration and
468
growth in LB medium for 48h at 37°C. Wells were stained with crystal violet (upper
469
panel). Quantification of biofilm formation was performed after crystal violet
470
extraction and measurement (OD620). Lower panel: normalized biofilm formation,
471
calculated as total biofilm (expressed as the OD620) divided by growth (expressed in
472
CFUs). Values are presented as mean ± standard deviation (SD) of three independent
473
experiments. Asterisks indicate: *, p<0.05; **, p<0.01.
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Supplementary Figure 1.
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Confocal scanning laser microscopy. Fluorescence (live/dead) expressed as percentage,
477
for each strain, at different time points.
478
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Supplementary Figure 2. Regrowth capacity estimated for A pittii populations
480
maintained on solid surfaces at time 0 or at different time points after desiccation.
481
Stressed populations were resuspended in LB medium, incubated for 48h at 37ºC and
482
the OD620 of bacterial cultures was measured. Columns, from white to black, strains 23
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LMG, HUMV 0315, HUMV 4336, HUMV 6207, HUMV 5918 and HUMV 6483. Values are
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presented as mean ± standard deviation (SD) of three independent experiments.
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