Effect of Relative Humidity on the Inactivation of Airborne Serratia marcescens by Ultraviolet Radiation

Apparatus was designed and constructed in which a bacterial aerosol of known age, particle size, and relative humidity (RH) could be exposed to ultraviolet (UV) radiation of measured intensity for a given period of time and then be sampled quantitatively. Aerosols of Serratia marcescens were exposed to UV dosages between 96.0 and 0.75 (muw-sec)/cm(2) at humidities ranging from 25 to 90%. A sharp decline in the fraction of organisms killed was found at RH values above 60 to 70%. Above 80% RH, there was evidence for reactivation induced by UV. The plot of "log fraction organisms remaining" versus UV dose was curvilinear, suggesting noncompliance with the monomolecular law of reaction velocity, but the Bunsen-Roscoe law of reciprocity between time and intensity of UV exposure was demonstrated to hold. These results could be accounted for by postulating the presence in the aerosol of two populations of organisms with different sensitivities to UV, each individually obeying the monomolecular law of reaction velocity. The data amplify existing information on the relationship between UV disinfection of airborne organisms and RH. In the middle range of humidities, the sensitivity of the organisms to UV was greater than would be expected from published reports.     

Cation transport in Serratia marcescens and Serratia marinorubra

The sodium, potassium, and magnesium ion contents of Serratia marcescens and those of its salt-tolerant relative, S. marinoruba, were determined by atomic-absorption spectrometry. The intracellular K(+) and Mg(2+) contents of both microorganisms were found to be dependent on the ionic strength of the growth or suspending medium. The Mg(2+) content of S. marinoruba was generally greater than that of S. marcescens. The Na(+) content of the cells was normally low and did not increase as the cells aged or when the cells were grown in media of high ionic strength. The transport of K(+) by resting cells suspended in hypertonic solution was studied by chemical and light-scattering techniques and was found to be more rapid in S. marcescens than in S. marinorubra. The slower rate of K(+) transport in S. marinorubra is probably related to the lower glycogen reserves found in resting cells of this microorganism. K(+) transport was found to have a pH optimum of 5.5 to 6.1 for S. marcescens, and the K(m) for K(+) was approximately 1.6 mm. Na(+) and Mg(2+) were not taken up by the cells, although the presence of Mg(2+) tended to decrease rates of K(+) uptake. Tris-(hydroxymethyl)aminomethane, routinely used for resuspending the cells, was apparently taken up by the cells at pH >7.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Role of Relative Humidity in the Survival of Airborne Mycoplasma pneumoniae

Aerosols of Mycoplasma pneumoniae were studied at several relative humidities at a controlled temperature of 27 C. Production of an experimentally reproducible aerosol required preatomization of the organism in its suspending fluid and was dependent on the type of fluid used in atomization as well as on the procedures used to produce an aerosol. The airborne particles studied were within the range of epidemiological significance, with most being 2 mum or less in diameter. Survival of the airborne mycoplasma in these particles was found to be best at very low and at very high humidities. The most lethal relative humidity levels were at 60 and 80%, at which levels fewer than 1% of the organisms survived over a 4-hr observation period. However, survival of the organism at most relative humidity levels was such that long-term infectivity could be expected from aerosols of M. pneumoniae. Because of the extreme sensitivity of M. pneumoniae at critical humidity levels, control of the airborne transmission of these organisms may be possible in selected spaces.     

Aerosol Survival of Pasteurella tularensis and the Influence of Relative Humidity

The aerosol survival in air was determined for Pasteurella tularensis live vaccine strain (LVS) as a function of relative humidity (RH). Three different preparations of bacteria were used: (i) liquid suspension of P. tularensis LVS in spent culture medium; (ii) powders of P. tularensis LVS freeze-dried in spent culture fluid; (iii) P. tularensis LVS freeze-dried in spent culture fluid and then reconstituted with distilled water and disseminated as a liquid suspension. Preparation (i) gave greatest survival at high RH and lowest survival at intermediate RH. Preparation (ii), in contrast, gave greatest survival at low RH and minimum survival at 81% RH. Preparation (iii) was the same as preparation (i), i.e., the process of freeze-drying and reconstituting with distilled water before aerosol formation had little or no effect upon aerosol survival as a function of RH. Hence, control of aerosol survival appears to be through the water content of P. tularensis LVS at the moment of aerosol generation rather than the water content of the bacteria in the aerosol phase.     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

The Effect of Relative Humidity on the Survival of Three Serotypes of Klebsiella

S ummary . Any factor which enhances the survival of human pathogens in the hospital environment can result in increased exposure of the patient to such pathogens and perhaps contribute to an increase in patient infections. Klebsiella serotypes 3, 9 and 69 were exposed on glass to relative humidities (R.H.) of 11, 33, 53 and 85%. Death rates for all serotypes, at a constant 25°, could be correlated with the amount of moisture in the air. Prolonged survival was demonstrated at 11 and 33% R.H. with an increased death rate at 53 and 85%. The implications of this phenomenon are discussed.     

Regulation of Stomatal Defense by Air Relative Humidity

It has long been observed that environmental conditions play crucial roles in modulating immunity and disease in plants and animals. For instance, many bacterial plant disease outbreaks occur after periods of high humidity and rain. A critical step in bacterial infection is entry into the plant interior through wounds and natural openings, such as stomata, which are adjustable microscopic pores in the epidermal tissue. Several studies have shown that stomatal closure is an integral part of the plant immune response to reduce pathogen invasion. In this study, we found that high humidity can effectively compromise Pseudomonas syringae-triggered stomatal closure in both Phaseolus vulgaris and Arabidopsis (Arabidopsis thaliana), which is accompanied by early up-regulation of the jasmonic acid (JA) pathway and simultaneous down-regulation of salicylic acid (SA) pathway in guard cells. Furthermore, SA-dependent response, but not JA-dependent response, is faster in guard cells than in whole leaves, suggesting that the SA signaling in guard cells may be independent from other cell types. Thus, we conclude that high humidity, a well-known disease-promoting environmental condition, acts in part by suppressing stomatal defense and is linked to hormone signaling in guard cells.The phyllosphere is one of the most diverse niches for microbe inhabitation. Numerous bacteria can survive and proliferate on the surface of the plant without causing any harm (Lindow and Brandl, 2003). However, for a bacterial pathogen to cause disease, it must penetrate through the plant epidermis and be able to survive and proliferate inside the plant. The mode and mechanism of penetration into the plant tissue is a critical step for infection, especially for bacterial pathogens that rely on natural openings and accidental wounds on the plant surface to colonize internal tissues (Misas-Villamil et al., 2013). Stomata are an example of such openings, providing one of the main routes through which the foliar pathogen Pseudomonas syringae transitions from avirulent epiphytic to virulent endophytic lifestyles (Melotto et al., 2008). This abundant opening in the epidermal tissue is not a passive port that allows unrestricted entry of microbes. It has been shown that plants are able to respond to human and plant bacterial pathogens by actively closing the stomatal pore (McDonald and Cahill, 1999; Melotto et al., 2006; Gudesblat et al., 2009; Zhang et al., 2010; Roy et al., 2013; Arnaud and Hwang, 2015), a phenomenon described as stomatal immunity (Sawinski et al., 2013). Several lines of evidence point to the complexity of this response and show that stomatal closure is an integral basal plant defense mechanism to restrict the invasion of pathogenic bacteria into plant tissues (Ali et al., 2007; Melotto et al., 2008; Zhang et al., 2008; Gudesblat et al., 2009). However, certain bacterial pathogens, such as Xanthomonas campestris pv campestris (Gudesblat et al., 2009), P. syringae pv syringae (Pss) B728a (Schellenberg et al., 2010), and P. syringae pvs tabaci, tomato, and maculicola (Melotto et al., 2006), can successfully cause disease by producing toxins that overcome stomatal immunity. Specifically, P. syringae pv tomato (Pst) DC3000 uses coronatine (COR) as such a toxin.In this study, we focused on elucidating environmental regulation of stomatal-based defense against bacterial invasion. Changes in environmental conditions, such as air relative humidity (RH), light, and carbon dioxide concentration regulate guard cell turgidity that consequently alters stomatal aperture size and the basic functions of stomata in plants, i.e. exchange of photosynthetic gases and regulation of water loss by transpiration (Zelitch, 1969; Schroeder et al., 2001; Fan et al., 2004). In natural conditions, plants are exposed to both biotic and abiotic stresses, and guard cells need to prioritize their response to the simultaneous occurrence of these stresses. For instance, it is a common observation that severe outbreaks of bacterial disease in the field are often associated with periods of heavy rain or high air humidity (Goode and Sasser, 1980). Mechanical wounding of plant tissues by rain might be one way that allows pathogens to bypass the stomatal route and gain unprecedented access to the plant interior. Additionally, the formation of large bacterial aggregates under high humidity on the leaf surface (Monier and Lindow, 2004) and splashing of bacteria during rain may also contribute to the spreading of disease at a higher rate. Interestingly, to ensure infection in the laboratory, researchers commonly expose plants to very high humidity for an extended period after surface inoculation. Here, we demonstrate that high RH compromises stomatal defense in Arabidopsis (Arabidopsis thaliana) and common bean (Phaseolus vulgaris) against P. syringae, allowing more bacteria to enter the leaf tissue and contributing to severe infections. Compromised bacterial-triggered stomatal closure due to high RH is accompanied by changes in plant hormone signaling in Arabidopsis. Specifically, high RH leads to activation of the jasmonic acid (JA) signaling pathway and down-regulation of the salicylic acid (SA) signaling in guard cells. These results connect plant physiology with epidemiology and advance the current understanding of foliar bacterial infection in plants.     

The constitutive K+ pump in Serratia marcescens

Transport of K+ and H+ in the anaeronically and aerobically grown bacterium Serratia marcescens has been studied. The volumes of one cell of the anaerobically and aerobically grown bacterium were 3.7 X 10(-13) cm3 and 2.4 X 10(-13) cm3, respectively. Irrespective of the growth conditions the bacteria manifested the same respiration rate. However, the values of membrane potential for the anaerobically and aerobically grown bacterium were different and equal to -130 mV and -175 mV (interior negative), respectively, in the absence of an exogenic energy source. KCN + DCCD decreases delta psi down to almost zero in both species. DCCD alone decreases delta psi partially in anaerobes and increases delta psi in aerobes, whereas KCN alone reduces delta psi partially in both species. The introduction of glucose into the medium containing K+ reduces the absolute value of delta psi to [-160] mV in aerobes and to [-20] mV in anaerobes. The effect is not observed without external K+. In the presence of arsenate a delta psi is not reduced after the addition of glucose. At pH 7.5-7.8 the ATP level in aerobes grows notably faster than in anaerobes. The H+ extrusion becomes intensified when K+ uptake is activated by the increase in external osmotic pressure. Apparent Km and Vmax for K+ accumulation are 1.2 mM and 0.4 mM.min-1.g-1. The decrease of delta psi by glucose or KCN + DCCD have no effect on the K+ uptake whereas CCCP inhibits potassium accumulation. At the same time, arsenate stabilizes the delta psi value, but blocks K+ uptake. The accumulation of K+ correlates with the potassium equilibrium potential of -200 mV calculated according to the Nernst equation, whereas the delta psi measured was not more than [-25] mV. The calculated H+/ATP stoichiometry was 3.3 for aerobes. It was assumed that a constitutive K+ pump having a K+/ATP ratio equal to 2 or 3 operates in S. marcescens membranes.     

Influence of Relative Humidity on the Survival of Some Airborne Viruses

A system for studying the effects of relative humidity (RH) and temperature on biological aerosols, utilizing a modified toroid for a static aerosol chamber, is described. Studies were conducted at 23 C and at three RH levels (10, 35, and 90%) with four viruses (Newcastle disease virus, infectious bovine rhinotracheitis virus, vesicular stomatitis virus, and Escherichia coli B T3 bacteriophage). Virus loss on aerosol generation was consistently lower at 90% than at 10 or 35% RH. When stored at 23 C, Newcastle disease virus and vesicular stomatitis virus survived best at 10% RH. Infectious bovine rhinotracheitis virus and E. coli B T3 bacteriophage survived storage at 23 C best at 90% RH.     

Effect of Relative Humidity on the Survival of Airborne Unicellular Algae

A method is described which is suitable for assessing the effects of relative humidity (RH) on the viability of two unicellular algae in experimental aerosols. Viable cells of Nannochloris atomus collected from the airborne state were detected by plating onto agar surfaces of an appropriate growth medium, whereas viable airborne cells of Synechococcus sp., because of unreliable growth on solid media, were determined by a liquid assay system. The assays were performed at intervals during short-term and prolonged storage of algal aerosols in chambers preconditioned to a selected RH and temperature. Both species showed the greatest loss in viability during the first minute after atomization, and the extent of this inactivation, as a function of RH, reflected the subsequent long-term survival. The airborne eukaryotic alga was unable to survive at an RH below 91%, whereas the airborne prokaryotic alga was comparatively stable over a wide humidity range. Initial inactivation was least and long-term survival best, for both species, at 94% RH.     

Pigmentation and Acriflavine Resistance in Serratia marcescens

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.