Survival of Yersinia pestis on environmental surfaces

The survival of two strains of Yersinia pestis (avirulent A1122 and virulent Harbin) on the surfaces of four materials was investigated. Viability was evaluated with epifluorescence microscopy by using the metabolic stain cyanoditolyl tetrazolium chloride and plate counts. Small numbers of cells suspended in phosphate buffer survived 2 to 4 h after visible drying on stainless steel, polyethylene, or glass and beyond 48 h on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 h on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 h on paper. These data suggest that Y. pestis maintains viability for extended periods (last measured at 5 days) under controlled conditions.     

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

AFM study of the colonisation of stainless steel by Aquabacterium commune

Aquabacterium commune, a member of the beta proteobacteria family that is a recently isolated, predominant member of various European drinking water distribution system biofilms, was selected as a test organism in this study. Attachment of A. commune cells onto two increasingly popular pipe materials, stainless steel EN1.4307 and medium density polyethylene (MDPE) was studied at 15 °C, 150 rpm, and a hydraulic retention time of 10.5 h in a rotating annular biofilm (RAB) reactor. Planktonic and sessile growth was monitored by spread plate technique. Atomic force microscopy (AFM) was used to obtain information about surface topography and biofilm formation pattern. Our study has shown that: (i) Steady-state conditions were reached after ca. 100 h for both materials; (ii) biofilm cell density on MDPE slides is four times greater than on stainless; (iii) the primary colonization of MDPE and stainless steel occurred at the edge of the slides; and (iv) no preferential attachment to stainless steel grain boundaries was observed. Stainless-steel manufacturers and suppliers, researchers, and companies working in the drinking-water sector will benefit from this paper. It is suggested that electropolishing of stainless-steel pipes for drinking water installations is not necessary to remove specific biofilm formation sites (i.e. grain boundaries). Furthermore, this paper provides, for the first time, some fundamental information for the continuous cultivation of the recently isolated drinking water microorganism, A. commune.     

The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces

Twelve graft copolymers have been evaluated for their ability to prevent the adhesion of bacteria to substrata. The copolymers had polyethylene glycol (PEG) side-chains (‘teeth’) and a backbone that was either uncharged, acidic, basic or amphoteric. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite substrata, and 2-hpetri-dish adhesion experiments performed with bacteria isolated from marine (Pseudomonas sp. NCMB 2021), paper mill (S. marcescens NCIB 12211) and oral (S. mutans NCTC 10449) environments. The copolymers containing the most charged groups in the backbone had the most significant effect on bacterial adhesion levels, with anti-adhesive effects up to 99% achieved. An amphoteric copolymer (Compound 12) on glass, and acidic copolymer (Compound 11) on stainless steel and hydroxyapatite gave the most impressive anti-adhesive effects. These copolymers had non-specific bacterial anti-adhesive properties.It is proposed that the graft copolymers adsorbed onto hydrophilic surfaces via their charged backbone in such a way that the PEG side-chains were pointing out into the aqueous phase, and it was this orientation that was responsible for the observed anti-adhesive effect.     

Laboratory studies on adhesion of microalgae to hard substrates

Adhesion of Chlorella vulgaris(chlorophyceae), Nitzschia amphibia(bacillariophceae) and Chroococcus minutus(cyanobacteria) to hydrophobic (perspex, titanium and stainless steel 316-L), hydrophilic (glass) and toxic (copper, aluminium brass and admiralty brass) substrata were studied in the laboratory. The influence of surface wettability, surface roughness, pH of the medium, culture age, culture density, cell viability and presence of organic and bacterial films on the adhesion of Nitzschia amphibia was also studied using titanium, stainless steel and glass surfaces. All three organisms attached more on titanium and stainless steel and less on copper and its alloys. The attachment varied significantly with respect to exposure time and different materials. The attachment was higher on rough surfaces when compared to smooth surfaces. Attachment was higher on pH 7 and above. The presence of organic film increased the attachment significantly when compared to control. The number of attached cells was found to be directly proportional to the culture density. Attachment by log phase cells was significantly higher when compared to stationary phase cells. Live cells attached more when compared to heat killed and formalin killed cells. Bacterial films of Pseudomonas putida increased the algal attachment significantly. %     

Vapour‐phase hydrogen peroxide inactivates Yersinia pestis dried on polymers,steel, and glass surfaces

Aims: This study evaluated the inactivation of virulent Yersinia pestis dried on polymers, steel, and glass surfaces using vapour‐phase hydrogen peroxide. Methods and Results: A suspension of Y. pestis CO92 (1·70 × 10⁸ CFU) was dried on 10 different types of test surfaces and exposed to vapour‐phase hydrogen peroxide fumigation for a contact time of 2 h. A significant reduction in the log₁₀ CFU of Y. pestis on all 10 materials was observed between the controls evaluated after a 1 h drying time and unexposed controls evaluated after the decontamination run. Qualitative growth assessment showed that vapour‐phase hydrogen peroxide exposure inactivated Y. pestis on all replicates of the 10 test materials as well as biological indicators up to 7 days postexposure. Conclusions: Virulent Y. pestis CO92 is inactivated on polymers, steel, and glass surfaces when exposed to vapour‐phase hydrogen peroxide without observable physical damage to the test materials. Significance and Impact of the Study: This study provides information for using vapour‐phase hydrogen peroxide as a practical process for the decontamination of virulent Y. pestis in circumstances where time‐dependent attenuation/inactivation or liquid/heat decontamination may not be the most suitable approach.     

Neutrophils are important in early control of lung infection by Yersinia pestis

In this paper we evaluate the role of neutrophils in pneumonic plague. Splenic neutrophils from naïve BALB/c mice were found to reduce numbers of culturable Yersinia pestis strain GB in suspension. A murine, BALB/c, intranasal model of pneumonic plague was used in conjunction with in vivo neutrophil ablation, using the GR-1 antibody. This treatment reduced neutrophil numbers without affecting other leukocyte numbers. Neutrophil ablated mice exhibited increased bacterial colonisation of the lung 24 h post infection. Furthermore, exposure of Y. pestis to human neutrophils resulted in a 5-fold reduction in the number of viable bacterial cells, whereas, PBMCs had no effect.     

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages. Received 16 December 1997/ Accepted in revised form 9 March 1998     

Dermal Neutrophil,Macrophage and Dendritic Cell Responses to Yersinia pestis Transmitted by Fleas

Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.     

Shewanella putrefaciens Adhesion and Biofilm Formation on Food Processing Surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10⁸ CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10² CFU/cm²) than in a batch system (reaching 10⁷ CFU/cm²), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

AFM study of the colonisation of stainless steel by Aquabacterium commune

Aquabacterium commune, a member of the beta proteobacteria family that is a recently isolated, predominant member of various European drinking water distribution system biofilms, was selected as a test organism in this study. Attachment of A. commune cells onto two increasingly popular pipe materials, stainless steel EN1.4307 and medium density polyethylene (MDPE) was studied at 15 °C, 150 rpm, and a hydraulic retention time of 10.5 h in a rotating annular biofilm (RAB) reactor. Planktonic and sessile growth was monitored by spread plate technique. Atomic force microscopy (AFM) was used to obtain information about surface topography and biofilm formation pattern. Our study has shown that: (i) Steady-state conditions were reached after ca. 100 h for both materials; (ii) biofilm cell density on MDPE slides is four times greater than on stainless; (iii) the primary colonization of MDPE and stainless steel occurred at the edge of the slides; and (iv) no preferential attachment to stainless steel grain boundaries was observed. Stainless-steel manufacturers and suppliers, researchers, and companies working in the drinking-water sector will benefit from this paper. It is suggested that electropolishing of stainless-steel pipes for drinking water installations is not necessary to remove specific biofilm formation sites (i.e. grain boundaries). Furthermore, this paper provides, for the first time, some fundamental information for the continuous cultivation of the recently isolated drinking water microorganism, A. commune.     

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real‐time PCR

Aim: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. Methods and Results: We evaluated the sample recovery efficiencies of two collection methods – swabs and wipes – for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real‐time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface‐dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Conclusions: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface‐dependent collection efficiencies observed with the virulent strains may arise from strain‐specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. Significance and Impact of the Study: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.     

Levan production by Zymomonas mobilis cells. Attached to plaited spheres

In this work, an immobilization method for polymer-levan production by a non-flocculating Z mobilis culture was developed. The extent of cell attachment to the stainless steel wire surface, culture growth and product synthesis were described. It was established that during short-term passive immobilization of non-flocculation Z mobilis cells on a stainless steel wire surface, sufficient amounts of biomass for proper levan and ethano fermentation could not be obtained. Adherence of cells was improved by pressing the paste-like biomass within stainless steel spheres knitted from wire with subsequent dehydration. Biomass fixed in metal spheres was used for repeated batch fermentation of levan. The activation period of cells within wire spheres (WS) was 48 h in duration. During this time, cell growth stabilized at production levels of ethanol and levan of Q_eth = 1.238 g/l × h and q_eth = 0.47 g/l × h; Q_eth = 0.526 g/l × h and q_eth = 0.20 g/l × h. Five stable fermentation cycles were realized using one wire sphere inoculum, and maintaining a stable ratio of 2.4 of biomass suspended in the medium to biomass fixed in the sphere. Using fixed Z mobilis biomass in the WS, the total amount of inoculum could be reduced for batch fermentation. Large plaited wire spheres with biomass may have potential in fermentation in viscous systems, including levan production.     

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Caspase-3 Mediates the Pathogenic Effect of Yersinia pestis YopM in Liver of C57BL/6 Mice and Contributes to YopM's Function in Spleen

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G⁺, F4/80⁺, and iNOS⁺ cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM''s action and supports the hypothesis that in liver YopM''s main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.     

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Background

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.

Methodology/Principal Findings

The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10³ CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.

Conclusions/Significance

Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.     

Physiology of biofilms of thermophilic bacilli—potential consequences for cleaning

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10^6.0 cells/cm² in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 °C developed a mature biofilm of >10^6.0 cells/cm² within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 m thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.     

Genotyping and Phylogenetic Analysis of Yersinia pestis by MLVA: Insights into the Worldwide Expansion of Central Asia Plague Foci

Background

The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar.

Methodology/Principal Findings

Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci.

Conclusions/Significance

The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an unprecedented view on the expansion and microevolution of Y. pestis.     

Different region analysis for genotyping Yersinia pestis isolates from China

Background

DFR (different region) analysis has been developed for typing Yesinia pestis in our previous study, and in this study, we extended this method by using 23 DFRs to investigate 909 Chinese Y. pestis strains for validating DFR-based genotyping method and better understanding adaptive microevolution of Y. pestis.

Methodology/Principal Findings

On the basis of PCR and Bionumerics data analysis, 909 Y. pestis strains were genotyped into 32 genomovars according to their DFR profiles. New terms, Major genomovar and Minor genomovar, were coined for illustrating evolutionary relationship between Y. pestis strains from different plague foci and different hosts. In silico DFR profiling of the completed or draft genomes shed lights on the evolutionary scenario of Y. pestis from Y. pseudotuberculosis. Notably, several sequenced Y. pestis strains share the same DFR profiles with Chinese strains, providing data for revealing the global plague foci expansion.

Conclusions/significance

Distribution of Y. pestis genomovars is plague focus-specific. Microevolution of biovar Orientalis was deduced according to DFR profiles. DFR analysis turns to be an efficient and inexpensive method to portrait the genome plasticity of Y. pestis based on horizontal gene transfer (HGT). DFR analysis can also be used as a tool in comparative and evolutionary genomic research for other bacteria with similar genome plasticity.     

Glass microspargers as effective frit spargers in single use bioreactors

For multiple-use bench scale and larger bioreactors, sintered stainless steel frit spargers are commonly used as microspargers. For bench-scale single-use bioreactors (SUBs), existing microspargers are sintered plastics, such as polyethylene. However, though plastics are readily sterilized by irradiation making them convenient for single use, these designs overlook surface energy properties of the materials of construction. For these sintered plastic spargers, forces at the water-air-surface interface cause bubble coalescence, leading to lower effective mass transfer, higher gas flow rates, and differing pCO₂ profiles in cell culture. Alternative materials of construction were evaluated based on contact angle information and bubble formation observations. Sintered glass was chosen over thermoplastic polymers for higher surface wettability as described in the glass/water contact angle, its history as a commonly sintered material, and availability at costs suitable for single use applications. Glass sintered spargers and traditional stainless steel frit spargers were compared by porosity, bubble size, and k_La studies. Mass transfer (k_La) and cell culture performance equal or greater than a standard 20 μm stainless steel microsparger mass transfer efficiency was achieved by a glass frit sparger, of international porosity standard “P40” according to ISO 4793-80, which corresponds to a range of 16–40 μm.