Growth Phase- and Desiccation-Dependent Acinetobacter baumannii Morphology: An Atomic Force Microscopy Investigation.

Acinetobacter baumannii has emerged as a major bacterial pathogen during the past three decades. The majority of the A. baumannii infections occur in hospitals and are caused by strains endowed with high desiccation tolerance, which represents an essential feature for the adaptation to the nosocomial environment. This work aims at investigating the desiccation response of the multidrug-resistant A. baumannii strain ACICU as a function of the bacterial growth phase and oxygen availability, by correlating bacterial survival with shape alterations. The three-dimensional morphological analysis of bacteria was carried out by atomic force microscopy (AFM), following the evolution of bacterial shape descriptors, such as the area, volume, roughness of individual cell membranes, and the cell cluster roughness, which exhibited peculiar and distinctive behavior as a function of the growth conditions. AFM images of A. baumannii ACICU cells revealed the prevalence of the coccoid morphology at all growth stages, with a tendency to reduce their size in the stationary phase, accompanied by a higher survival rate to air-drying. Moreover, cells harvested from the logarithmic phase featured a larger volume and resulted to be more sensitive to desiccation compared to the cells harvested at later growth stages. In addition, oxygen deprivation caused a significant decrease in cellular size and was associated with the formation of pores in the cell membrane, accompanied by a relative reduction in culturability after desiccation. Morphological plasticity and multidrug resistance may contribute to desiccation tolerance and therefore to persistence in the hospital setting.

New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors.

The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model.IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

Demonstration of PAM4-OCDM system with electrical amplitude-level pre-tuning and post-equalization for data centers applications.

A PAM4-OCDM system with optical multi-/demultiplexing and electrical pre-/post-processing is proposed for short-reach applications. We experimentally demonstrate, for the first time, a power-efficient 4 OC x 10 GSymbol/s PAM4-OCDM system. The four PSK-OCs are simultaneously generated using a single light source and a passive multiport optical encoder and received by a single optical decoder and cascaded DSP. The effectiveness of the electrical-domain amplitude level pre-tuning and post-equalizer are demonstrated, considering different values of shot and beat noises.

All-optical Nyquist-OTDM to Nyquist-WDM conversion for high-granular flexible optical networks.

We have proposed and experimentally demonstrated all-optical multiplexing (MUX)-format conversion from Nyquist optical time division multiplex (OTDM) to Nyquist wavelength division multiplex (WDM). The system is simply configured with a straight-type phase modulator (PM) driven by a sinusoidal wave and an optical Nyquist filter. In the theoretical investigation, it is proved that the single Nyquist signal is completely converted to Nyquist WDM signal, which consists of two half-baud-rate signals with different carrier frequencies. The theoretical modulation voltage for the phase modulator is slightly lower than Vπ: 0.913 Vπ, and it is experimentally verified. In the experimental demonstrations, 50 Gbaud to 25 Gbaud x 2 and 25 Gbaud to 12.5 Gbaud x 2 conversions are successfully demonstrated with quite low optical signal-to-noise ratio (OSNR) penalties. In addition, cascaded MUX-format conversion is also demonstrated; 50 Gbaud Nyquist signal is converted to four channels of 12.5 Gbaud Nyquist signals.

Chromatic dispersion monitoring and adaptive compensation using pilot symbols in an 8 x 12.5 Gbit/s all-optical OFDM system.

We propose and experimentally demonstrate a novel technique for chromatic dispersion (CD) monitoring and adaptive compensation in an 8 x 12.5 Gbit/s all-optical orthogonal frequency-division multiplexing (AO-OFDM) system by using two pilot symbols and a virtually imaged phased array (VIPA) for a tunable CD compensator. The two pilot symbols are added to the first and the last sub-channels of the OFDM signal, and their relative time delay is detected and used for CD estimation at the CD monitoring circuit. The monitored CD value is fed to VIPA for CD compensation. In the experiments, the relative time delay between the two pilot symbols was successfully observed, and the adaptive CD compensation drastically improved the bit-error-rate (BER) from over 10(-5) to under 10(-9). The estimated CD values showed less than 10 ps/nm difference from the values measured by a photonic dispersion analyzer, which is accurate enough since the AO-OFDM system can keep BER<10(-9) upto 20 ps/nm residual CD.

A low-power photonic quantization approach using OFDM subcarrier spectral shifts.

Photonic analog-to-digital conversion and optical quantization are demonstrated, based on the spectral shifts of orthogonal frequency division multiplexing subcarriers and a frequency-packed arrayed waveguide grating. The system is extremely low-energy consuming since the spectral shifts are small and generated by cross-phase modulation, using a linear-slope high-speed and low-jitter pulse train generated by a mode locked laser diode. The feasibility of a 2, 3 and 4-bit optical quantization scheme is demonstrated.

First demonstration of a scalable MDM/CDM optical access system.

A novel hybrid all-optical mode-division multiplexing and code division multiplexing architecture for flexible and scalable access networks is presented. We successfully demonstrate, for the first time, an asynchronous on-off keying modulation, 2 mode x 4 code x 10 Gbps transmission over 42-km link, using a set of single-mode and two-mode fibers, without dispersion compensation. The four phase-shift keyed optical codes are generated at a single wavelength, by a multiport encoder/decoder, and we use an optical mode multiplexer/demultiplexer in the remote node and at the central office. We also experimentally evaluate the mode crosstalk tolerance considering different access span distances for the LP(01) and LP(11) modes.

Tunable optical code converter using XPM and linear-slope pulse streams generated by FBGs.

We propose for the first time (to the best of our knowledge) and demonstrate a tunable multiple optical code (OC) converter for flexible networks that allows dynamic code (bandwidth) allocation in a way similar to a tunable laser. With respect to other OC converters, this scheme can convert both specific and multiple OCs by changing only the amplifier gain. The proposed scheme uses cross-phase modulation (XPM) and two linear-slope control pulse streams generated by fiber Bragg gratings (FBGs) in the C-band. The OC converter can be used to avoid collisions in optical packet switching networks, and we analyze the corresponding packet loss probability performance.

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging.

IMPORTANCE: By harnessing the versatility of fluorescence microscopy and super-resolution imaging, bacteriologists explore critical aspects of bacterial physiology and resolve bacterial structures sized beyond the light diffraction limit. These techniques are based on fluorophores with profitable photochemical and tagging properties. The paucity of available far-red (FR)-emitting dyes for bacterial imaging strongly limits the multicolor choice of bacteriologists, hindering the possibility of labeling multiple structures in a single experiment. The set of FR fluorophores characterized in this study expands the palette of dyes useful for microbiologists, as they can be used for bacterial LIVE/DEAD staining and for tagging the membranes of viable Escherichia coli and Bacillus subtilis cells. The absence of toxicity makes these dyes suitable for live-cell imaging and allows monitoring of bacterial membrane biogenesis. Moreover, a newly synthesized FR-fluorophore can be employed for imaging bacterial membranes with stimulated emission depletion microscopy, a super-resolution technique capable of increasing the resolving power of conventional microscopes.

An image processing-based quantification of gram variability in Acinetobacter baumannii.

Gram staining differentiates bacteria as gram-positive and gram-negative, depending on their cell wall constituents, and coloring cells in violet and pink, respectively. Sometimes, a subpopulation of the same bacterial species assumes different colors, ranging from pink to violet, for reasons that are not completely understood yet. We analyze conventional brightfield images and use an automated pipeline to count pink and violet cells. The ImageJ-based processing algorithm quantifies the gram variability in Acinetobacter baumannii ACICU in the stationary phase of growth with a percentage of 66% pink cells. Different bacterial strains and cell growth stages have been considered. RESEARCH HIGHLIGHTS: Gram staining differentiates bacteria into gram-positive (violet) and gram-negative (pink). Gram variability represents an inhomogeneous distribution of pink and violet cells within the same species. We developed an ImageJ-based pipeline for the quantification of Gram variability in Acinetobacter baumannii.

What else can an AWG do?

The present paper aims to describe other functionalities for an arrayed waveguide grating (AWG)-based device, showing that this widely used configuration can be designed not only to frequency multiplex/demultiplex wavelength division multiplexing (WDM) signals, but also to perform the discrete Fourier transform (DFT) and the discrete fractional Fourier transform (DFrFT) of a signal, in all-optical orthogonal frequency division multiplexing (OFDM) systems. In addition 1 × N and N × N phased array switches architectures are described, as well as a new configuration to perform polarization diversity demultiplexing. Finally, a general approach, based on an analogy with the finite impulse response (FIR) filter approach, is presented to design optical modulators for any modulation format, using either phase modulators (PM) or electro-absorption modulators (EAM).

Demonstration and performance investigation of all-optical OFDM systems based on arrayed waveguide gratings.

We experimentally demonstrate an 8 x 12.5 Gbit/s all-optical orthogonal frequency-division multiplexing (AO-OFDM) system using arrayed waveguide gratings (AWGs), which perform discrete Fourier transform (DFT) and inverse DFT (IDFT) of a signal directly in the optical domain. The experimental results show that frequency orthogonality of OFDM sub-channels is degraded in the AWG due to the slab-diffraction effect. To restore the frequency orthogonality and improve the system performance, we propose and demonstrate a waveform reshaping scheme, that improve the bit-error-rate (BER) from 10(-4) to 10(-6). We also experimentally investigate the influence of frequency mismatch between the OFDM signal and AWG at the receiver. The measured BER shows a serious degradation from 10(-6) to 10(-4) in case of ± 1.88 GHz frequency mismatch. To keep the BER under 10(-5), the frequency mismatch should be smaller than ± 0.5 GHz ( ± 4% of the channel spacing).

Noise suppression using optimum filtering of OCs generated by a multiport encoder/decoder.

We propose a novel receiver configuration using an extreme narrow band-optical band pass filter (ENB-OBPF) to reduce the multiple access interference (MAI) and beat noises in an optical code division multiplexing (OCDM) transmission. We numerically and experimentally demonstrate an enhancement of the code detectability, that allows us to increase the number of users in a passive optical network (PON) from 4 to 8 without any forward error correction (FEC).

Demonstration of asynchronous, 40 Gbps x 4-user DPSK-OCDMA transmission using a multi-port encoder/decoder.

We have developed a new 8-chip, 320 Gchip/s encoder/decoder with eight input/output ports, that can be used in 40-Gb/s PON networks. The device has been to multiplex four asynchronous 40 Gb/s users, using DPSK modulation. The transmission over 50 km has been successfully demonstrated for the first time.

Generalized fiber Fourier optics.

A twofold generalization of the optical schemes that perform the discrete Fourier transform (DFT) is given: new passive planar architectures are presented where the 2 × 2 3 dB couplers are replaced by M × M hybrids, reducing the number of required connections and phase shifters. Furthermore, the planar implementation of the discrete fractional Fourier transform (DFrFT) is also described, with a waveguide grating router (WGR) configuration and a properly modified slab coupler.

High-security 2.5 Gbps, polarization multiplexed 256-ary OCDM using a single multi-port encoder/decoder.

A block-ciphered (M-ary) optical code division multiplexing (OCDM) can provide larger security than a conventional OCDM system based on bit ciphering. We propose a polarization multiplexed (POL-MUX) M-ary OCDM system and demonstrated 2.5 Gbps, POL-MUX 256 ( = 16X16)-ary OCDM transmission using a single multi-port optical encoder/decoder (E/D). We show that this architecture reduces the number of required optical codes and enhances the system confidentiality.

STED nanoscopy of KK114-stained pathogenic bacteria.

Super-resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine-like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram-positive and Gram-negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114-staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells.

SSNOMBACTER: A collection of scattering-type scanning near-field optical microscopy and atomic force microscopy images of bacterial cells.

BACKGROUND: In recent years, a variety of imaging techniques operating at nanoscale resolution have been reported. These techniques have the potential to enrich our understanding of bacterial species relevant to human health, such as antibiotic-resistant pathogens. However, owing to the novelty of these techniques, their use is still confined to addressing very particular applications, and their availability is limited owing to associated costs and required expertise. Among these, scattering-type scanning near field optical microscopy (s-SNOM) has been demonstrated as a powerful tool for exploring important optical properties at nanoscale resolution, depending only on the size of a sharp tip. Despite its huge potential to resolve aspects that cannot be tackled otherwise, the penetration of s-SNOM into the life sciences is still proceeding at a slow pace for the aforementioned reasons. RESULTS: In this work we introduce SSNOMBACTER, a set of s-SNOM images collected on 15 bacterial species. These come accompanied by registered Atomic Force Microscopy images, which are useful for placing nanoscale optical information in a relevant topographic context. CONCLUSIONS: The proposed dataset aims to augment the popularity of s-SNOM and for accelerating its penetration in life sciences. Furthermore, we consider this dataset to be useful for the development and benchmarking of image analysis tools dedicated to s-SNOM imaging, which are scarce, despite the high need. In this latter context we discuss a series of image processing and analysis applications where SSNOMBACTER could be of help.

Geometrical-optics approach to measure the optical density of bacterial cultures using a LED-based photometer.

We develop a suitable geometrical-optics approach and demonstrate that it is possible to measure the optical density (OD) of bacterial cultures using a light emitting diode (LED)-based photometer. We measure both attenuation and spot-size variation, and we compensate for diffraction and stray-light impairment related to the incoherent source and large detection area. The approach is validated for different concentrations of two bacterial species, Escherichia coli and Staphylococcus aureus, that present different shapes and clustering organization.

Geometrical-optics approach to increase the accuracy in LED-based photometers for point-of-care testing.

A geometrical-optics approach is proposed to increase the accuracy in photometric measurements, using a point-of-care testing (POCT) LED-based sensor. Due to stray-light effects, the measurement accuracy depends on the dimension of the CMOS area, where the radiation is detected. We propose two image processing approaches and evaluate the influence of the sensor area. In addition, we demonstrate that with the same measurement, both absorption coefficient and refractive index can be determined, measuring the beam attenuation and the spot-size enlargement due to ray refraction.