Postantibiotic effects of subinhibitory concentrations of some antibiotics and their influence onPseudomonas aeruginosa enzymic activity

The postantibiotic effects of subinhibitory concentrations (PA SMEs) and virulence factor alterations induced by ciprofloxacin, tobramycin and netilmicin inPseudomonas aeruginosa were studied. After induction of the postantibiotic phase (PA) (2x or 4x MIC) the cultures were exposed to subinhibitory concentrations (0.1, 0.2 and 0.3x MIC) of the same antibiotic PA SME). The regrowth of treated as well as control cultures was followed for 24 or 45 h. In the sterile culture filtrates obtained from these bacterial cultures, elastase and proteinase were determined. Ciprofloxacin and aminoglycosides exhibited PA SMEs of 3.5–35 h for certain combinations of supra-subinhibitory antibiotic concentrations. Longer PA SMEs were observed after treatment with higher sub-MICs. Tobramycin at 0.2 and 0.3x MIC (postantibiotic phase induced by 2x MIC) and at all sub-MICs added to the bacteria previously exposed to 4x MIC do not allow any regrowth of bacterial culture. PA SMEs of tested antibiotics affected virulence factors ofP. aeruginosa. Elastase compared to proteinase was suppressed more effectively. Ciprofloxacin at 0.3x MIC reduced elastase and proteinase activity most significantly (to 14.2 and 60 % of the control values).     

Influence of the postantibiotic effect and postantibiotic Sub-MICs effect of netilmicin,tobramycin, ciprofloxacin and pefloxacin on alginate production byPseudomonas aeruginosa

The postantibiotic effect (PAE) (postantibiotic phase induced by 2× or 4×MIC) as well as the postantibiotic effect of subinhibitory concentrations (0.1×, 0.2× and 0.3× MIC) (PA SME) of netilmicin, tobramycin, ciprofloxacin and pefloxacin affected the production of the virulence factor alginate by aP. aeruginosa strain. Aminoglycosides and ciprofloxacin at a concentration of 4× MIC inhibited the alginate production more significantly than 2× MIC. Suprainhibitory concentrations of aminoglycosides were more effective than pefloxacin (2× or 4× MIC) and ciprofloxacin (2× MIC). PA SME demonstrated by the above antibiotics (with the exception of ciprofloxacin 2× MIC +00.1× MIC) suppressed alginate production more efficiently.     

Postantibiotic effects of imipenem and enoxacin againstS. typhimurium andS. enteritidis and the influence on their surface hydrophobicity

The influence of the postantibiotic effect (PAE) and the postantibiotic sub-MICs effect (PA SME) of imipenem and enoxacin on the surface hydrophobicity ofS. typhimurium andS. enteritidis strains were studied by evaluating Congo red binding and the aggregation in molar solutions of ammonium sulfate (SAT). A PAE was induced by 2× and 4× MIC of antibiotics tested for 0.5 h. Suprainhibitory concentrations of imipenem againstS. typhimurium induced a short PAE (0.3–0.6 h) compared toS. enteritidis (6.0–9.7 h). Suprasubinhibitory concentrations of imipenem did not allow a regrowth ofS. enteritidis. Similar results were also found for enoxacin. Evaluation of surface hydrophobic properties of the salmonellas after affecting both PAEs and PA SMEs has shown that imipenem at concentrations 4×MIC and 4×MIC+0.3×MIC partially influenced the hydrophobicity ofS. typhimurium. S. enteritidis was more susceptible toward both antibiotics tested.     

Inhibition ofPseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics

The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of quinolones (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin), aminoglycosides (amikacin, gentamicin, netilmicin, streptomycin, tobramycin), β-lactams (aztreonam, ceftazidime, imipenem, ticarcilin) and macrolides (erythromycin, roxitromycin) on the excretion of alginate by aP. aeruginosa strain were studied. Both β-lactam and macrolide antibiotics were found ineffective at the concentrations tested, except erythromycin and imipenem at 1/4 MIC. Aminoglycosides at a concentration of 1/4 MIC reduced most effectively the excretion of alginate. Quinolones were also effective at this sub-MIC; 1/16 MIC was ineffective with all antibiotics or stimulated the production of alginate.     

Postantibiotic effects of gentamicin and netilmicin onSerratia marcescens: Effects on hydrophobicity and motility

The impact of postantibiotic effect (PAE) of aminoglycosides (gentamicin, netilmicin) on cell-surface hydrophobicity and motility of a clinical isolateSerratia marcescens was evaluated. For the induction of PAE 2× and 4×MIC concentrations of both antibiotics were used. Gentamicin and netilmicin induced a PAE of similar duration after 2×MIC concentration (2.7 and 2.8 h, respectively). Both aminoglycosides demonstrated concentration-dependent PAE. At a concentration of 4×MIC they produced PAEs of 5.9 and 8.2h, respectively. The evaluation of hydrophobic properties ofS. marcescens after affecting PAE showed that both aminoglycosides inhibited adherence to xylene. This inhibition was also concentration-dependent. More expressive, was netilmicin which inhibited the adhesion by 70.5% at 2×MIC and by 85.2% at 4×MIC. Netilmicin inhibited also the adhesion to nitrocellulose filter by 34.7% at 4×MIC. Exposure of the bacterial cells to suprainhibitory concentrations of both aminoglycosides resulted only in moderate inhibition of motility of strain tested compared to the unexposed cells.     

Influence onEnterobacter cloacae metabolism, cell-surface hydrophobicity and motility of suprainhibitory concentrations of carbapenems

The impact of postantibiotic effect (PAE) of carbapenems (imipenem, meropenem) on the metabolism (biosynthesis of macromolecules, respiration), cell-surface hydrophobicity and motility of a clinical isolate ofEnterobacter cloacae was examined. The metabolism was evaluated after 16 h and after 1 d of cultivation using 2× and 4× minimum inhibitory concentrations (MIC) of both antibiotics for the induction of PAE. Imipenem at 4×MIC did not induce PAE. After a 16-h cultivation (in the postantibiotic phase of both carbapenems), inhibition of nucleosynthesis and protein synthesis was found; after a 1-d cultivation, during regrowth stimulation of mainly¹⁴C-leucine incorporation was found. The presence of the exogeneous intermediates of citrate cycle,viz. 2-oxoglutarate, increased the respiratory activity of the cells. The cell-surface hydrophobicity (evaluated by three methods—bacterial adhesion to hydrocarbon, nitrocellulose-filter test and salt-aggregation test) decreased after PAE of both carbapenems; meropenem was more effective. Motility (an important virulence factor) was inhibited in the postantibiotic phase of both carbapenems; the 4×MIC caused a higher inhibition.     

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties ofSalmonella strains

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied forSalmonella typhimurium andS. enteritidis strains. PAE was induced by 2× and 4×MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenicS. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed withS. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4×MIC of amikacin withS. enteritidis (6.9 h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASME's were different while concentrations of 2×MIC+0.2×MIC and 0.3×MIC, and of 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin did not allow any regrowth ofS. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by bothSalmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenicS. typhimurium strain. We noted the influence of Congo red binding after applying 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin forS. typhinurium and 2×MIC+0.1×MIC forS. enteritidis.     

Norfloxacin and ursolic acid: in vitro association and postantibiotic effect against Staphylococcus aureus

Aims: We investigated the effectiveness in vitro of the association between norfloxacin (NOR) and ursolic acid (UA) against Staphylococcus aureus. Methods and Results: The minimal inhibitory concentrations (MICs), the minimal bactericidal concentrations, the bacterial killing and the postantibiotic effect (PAE) of NOR and UA were determined both singly and in combination. A synergistic interaction was observed against Staph. aureus ATCC 29213: the mean PAEs were 3 h for NOR, ?1·2 h for UA (1 × MIC) and 2·0 h for UA (2 × MIC). Synergism was observed with longer PAEs and postantibiotic sub‐MIC effects after NOR/UA exposure. UA was also active against clinical isolates and methicillin‐resistant Staph. aureus. Conclusions: The application of antimicrobial combinations may address the rising resistance to established classes of both systemic and topical agents. Significance and Impact of the Study: In vitro interactions between NOR and UA may contribute to the development of novel topical agents for the treatment of skin infections as well as for topical formulations.     

Regulation of Caenorhabditis elegans and Pseudomonas aeruginosa machinery during interactions

The amenability of Caenorhabditis elegans against pathogen provides a valuable tool for studying host–pathogen interactions. Physiological experiments revealed that the P. aeruginosa was able to kill C. elegans efficiently. The effects of P. aeruginosa PA14, PAO1 and their isolated lipopolysaccharide (LPS) on the host system were analyzed. The LPS at higher concentrations (≥2 mg/ml) was toxic to the host animals. Kinetic studies using qPCR revealed the regulation of host-specific candidate antimicrobial genes during pathogen-mediated infections. In addition, the pathogen-specific virulent gene, exoT expression, was anlyzed and found to be varied during the interactions with the host system. Ability of the pathogens to modify their internal machinery in the presence of the host was analyzed by XRD, FTIR and PCA. LPS isolated from pathogens upon exposure to C. elegans showed modifications at their functional regions. LPS from PAO1 showed difference in d-spacing angle (Å) and °2Th position. FTIR spectra revealed alterations in polysaccharide (1,200–900 cm⁻¹) and fatty acid (3,000–2,800 cm⁻¹) regions of LPS from P. aeruginosa PAO1 exposed to the host system. These data provide additional insights on how the pathogens subvert its own and host machinery during interactions.     

Bioenergetic Investigation of the Effects of La(III) and Ca(II) on the Metabolic Activity of Tetrahymena thermophila BF5

The heat flux of Tetrahymena thermophila BF5 during growth and the effects of La³⁺ and Ca²⁺ on them were investigated with microcalorimetry; simultaneously, morphological changes of T. thermophila were obtained by light microscope. La³⁺ in low concentration (0–5.0 × 10^–4 mol/l) remarkably stimulated T. thermophila metabolism, but high dose of La³⁺ (5.8–8.6 × 10^–4 mol/l) restrained it in a linear manner with IC₅₀ being 7.2 × 10^–4 mol/l. In contrast, low concentration of Ca²⁺ did not manifest obvious stimulation on T. thermophila metabolism; moreover, the IC₅₀ of Ca²⁺ was much higher than that of La³⁺. Low concentration of La³⁺ did not lead to changes in appearance of T. thermophila, but low dose of Ca²⁺ clearly promoted the cell proliferation. In addition, the morphological changes of T. thermophila evoked by high concentrations of La³⁺ and Ca²⁺ were consistent with relevant microcalorimetric results. It is concluded that La and Ca influence T. thermophila via different pathways, and La represents toxic action rather than Ca analogy.     

Dynamics of cyanophage-like particles and algicidal bacteria causing Microcystis aeruginosa mortality

The dynamics of cyanophage-like particles and algicidal bacteria that infect the bloom-forming cyanobacterium Microcystis aeruginosa was followed in a hyper-eutrophic pond from September 1998 to August 1999. The densities of M. aeruginosa ranged between 4.0 × 10⁵ and 1.9 × 10⁷ cells ml⁻¹, whereas those of algicidal bacteria were between 4.0 and 5.1 × 10² plaque-forming units (PFU) ml⁻¹ and those of cyanophage-like particles were between <5.0 × 10² and 7.1 × 10³ PFU ml⁻¹. A significant relationship was found between the densities of algicidal bacteria and M. aeruginosa (r = 0.81, n = 69, P < 0.001), suggesting that the dynamics of the algicidal bacteria may regulate the abundance of M. aeruginosa. Occasional peaks of density of cyanophage-like particles were detected in October, June, and August, when sharp declines in M. aeruginosa cell densities were also observed. The densities of cyanophage-like particles became undetectable when the abundance of M. aeruginosa was low, suggesting the density-dependent infection of M. aeruginosa by cyanophage-like particles. Thus, we suggest that infections of both algicidal bacteria and cyanophage-like particles are important biological agents that decompose blooms of M. aeruginosa in freshwater environments. Received: August 31, 2000 / Accepted: December 6, 2000     

Antimicrobial efficacy of quaternary bisammonium salts and the effect of their sub-MICs onPseudomonas aeruginosa virulence factors

Antipseudomonadal activity of homologous series of six quaternary bisammonium salts (QBAS) (4,7-dioxo-3,8-dioxadekan-1,1-[bis(alkyldimethyldiammonium dibromide)] as well as the effect of their subinhibitory concentrations (sub-MICs) onPseudomonas aeruginosa virulence factors was studied. Antibacterial activity of QBAS increased up to a certain length of the chain and then decreased with further elongation. All the tested sub-MICs of QBAS caused a significant suppression of phospholipase C activity (to 0–41%). Elastase and proteinase activity were less efficiently reduced. A more effective decrease of these activities was only found after treatment with one-fourth of the MICs of the tested substances. QBAS caused only an erratic decrease of alginate production.     

Microbial communities in saline lakes of the Vestfold Hills (eastern Antarctica)

A detailed survey was undertaken of the microbial communities of 16 saline lakes in the Vestfold Hills (Princess Elizabeth Land, eastern Antarctica), which ranged in salinity from slightly brackish (4–5‰) to hypersaline (maximum: 174‰). Temperatures at comparable sampling depths in the lakes ranged from −12.2°C to +10.5°C. Ranges in the abundances of bacteria, heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were 1.40 × 10⁷ l⁻¹–1.58 × 10¹⁰ l⁻¹, 4.83 × 10⁴ l⁻¹–1.70 × 10⁷ l⁻¹ and 0–1.02 × 10⁷ l⁻¹, respectively. There was considerable variation across the salinity spectrum, though in the case of bacteria and PNAN significantly higher concentrations of cells were seen in two of the most saline lakes. The autotrophic ciliate Mesodinium rubrum occurred in all but five of the lakes and was found at salinity levels up to 108‰. Heterotrophic ciliates were generally scarce. Dinoflagellates, particularly Gonyaulax c.f. tamarensis, Gyrodinium lachryma and Gymnodinium sp., occurred in the majority of the lakes. On the basis of chlorophyll a concentrations, nutrient levels and microplankton concentrations the lakes spanned the spectrum from ultra-oligotrophic to oligo/mesotrophic. The most saline lakes had much reduced species diversity compared with the less saline environments. Isolation from the marine environment has led to nutrient depletion, simplification and a truncated trophic structure. Received: 19 September 1996 / Accepted: 13 January 1997     

Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1

According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 μg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70–74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat.     

Isolation and Characterization of Acetate-Utilizing Anaerobes from a Freshwater Sediment

Acetate-degrading anaerobic microorganisms in freshwater sediment were quantified by the most probable number technique. From the highest dilutions a methanogenic, a sulfate-reducing, and a nitrate-reducing microorganism were isolated with acetate as substrate. The methanogen (culture AMPB-Zg) was non-motile and rod-shaped with blunted ends (0.5–1 μm × 3–4 μm long). Doubling times with acetate at 30–35°C were 5.6–8.1 days. The methanogen grew only on acetate. Analysis of the 16S rRNA sequence showed that AMPB-Zg is closely related toMethanosaeta concilii. The isolated sulfate-reducing bacterium (strain ASRB-Zg) was rod-shaped with pointed ends (0.5–0.7 μm × 1.5–3.5 μm long), weakly motile, spore forming, and gram positive. At the optimum growth temperature of 30°C the doubling times with acetate were 3.9–5.3 days. The bacterium grew on a range of organic acids, such as acetate, butyrate, fumarate, and benzoate, but did not grow autotrophically with H₂, CO₂, and sulfate. The closest relative of strain ASRB-Zg isDesulfotomaculum acetoxidans. The nitrate-reducing bacterium (strain ANRB-Zg) was rod-shaped (0.5–0.7 μm × 0.7–1 μm long), weakly motile, and gram negative. Optimum growth with acetate occurred at 20–25°C. The bacterium grew on a range of organic substrates, such as acetate, butyrate, lactate, and glucose, and did grow autotrophically with H₂, CO₂, and oxygen but not with nitrate. In the presence of acetate and nitrate, thiosulfate was oxidized to sulfate. Phylogenetically, the closest relative of strain ANRB-Zg isVariovorax paradoxus.     

Bioelectrical activity in the heart of the lugworm Arenicola marina

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (−58.4 ± 3.2 mV), the average amplitude of the action potential (28.7 ± 4.7 mV) and the average total duration of the action potential (2,434 ± 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 × 10⁻⁸–5 × 10⁻⁵ M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 × 10⁻⁵ M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10⁻⁸–10⁻⁶ M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 × 10⁻⁶ M atropine. d-Tubocurarine (5 × 10⁻⁵ M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10⁻⁸–10⁻⁶ M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10⁻⁶ M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the β-blocker propranolol (5 × 10⁻⁶ M). The β-agonist isoproterenol (10⁻⁷–10⁻⁵ M) and the α-agonist xylometazoline (10⁻⁶–10⁻⁵ M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.     

Accelerated Biodegradation of High and Low Concentrations of p-Nitrophenol (PNP) by Bacterial Inoculation in Industrial Wastewater: The Role of Inoculum Size on Acclimation Period

The effect of inoculum size on the acclimation period and rate and extent of p-nitrophenol (PNP) degradation at high (1–10 mg/L) and low (26 μg/L) concentrations for two bacteria was determined in defined media as well as industrial wastewater. Increased inoculum size did not affect the acclimation period of either bacterium at high (1–10 mg/L) PNP concentrations. At low PNP concentrations (26 μg/L), the two bacteria behaved differently. The acclimation period was shortened and both the rate and extent of mineralization of PNP were enhanced by increasing the Corynebacterium sp. inoculum size from 3 × 10⁵ to 3 × 10⁶ cells/ml. Addition of phosphate or elimination of predators also reduced the acclimation period. Conversely, increasing the inoculum size from 3 × 10⁵ to 5 × 10⁶ cells/ml of Pseudomonas putida lengthened the acclimation period and reduced both the rate and extent of mineralization. It is suggested that, in a given environment, the success of an introduced species to enhance the degradation of a chemical depends upon (i) concentration of the chemical, (ii) selection of an appropriate microorganism, and (iii) utilization of a suitable inoculum size. Received: 1 April 1996 / Accepted: 6 May 1996     

BVPaP-3, a T7-Like Lytic Phage of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Its Isolation and Characterisation

The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58–59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6–10) and temperatures (4–40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10⁻⁹. Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.     

Interfacial Tension of the Lipid Membrane Formed from Phosphatidylcholine–Decanoic Acid and Phosphatidylcholine–Decylamine Systems

Interfacial tension has been determined for phosphatidylcholine (PC)–decanoic acid (DA) and PC–decylamine (DE) membranes. PC (lecithin), DA and DE were used in the experiments; the interfacial tension values of the pure components are 1.62 × 10⁻³, −2.38 × 10⁻² and −3.88 × 10⁻² N/m (hypothetical values for DA and DE), respectively. The 1:1 complexes were formed during formation of PC–DA and PC–DE membranes. The following parameters describing the complexes were determined: the surface concentrations of the lipid membranes formed from these complexes, A₃ ^{- 1} A_{3}^{ - 1} ; the interfacial tensions of such membranes, γ ₃; and the stability constants of these complexes, K.     

Asymbiotic germination of ornamental <Emphasis Type="Italic">Vanda:</Emphasis> in vitro germination and development of three hybrids

The majority of potted flowering orchids sold in the United States are Phalaenopsis hybrids. However, demand for other genera will undoubtedly increase as consumers become accustomed to growing orchids in the home. The objective of this study was to compare the germination and development of three Vanda hybrids, a genus with mass market potential. Specifically, we evaluated whether hybrids with different pedigrees have significantly different culture requirements. After 12 weeks culture, differences in germination and development were found among the three hybrids screened. Generally, seeds of Vanda Motes Primrose × Ascocenda Tavivat (hybrid S014) showed higher germination (82.0–95.3%) than seeds of V. Paki × (V. tessellata × V. cristata; hybrid S005) or (V. Joan Warne × V. Paki) × V. Loke (hybrid S013) when cultured on Knudson C, half-strength Murashige & Skoog, or PhytoTechnology Orchid Seed Sowing Medium under three photoperiods (8/16 h, 12/12 h, 16/12 h; light/dark). Protocorms of S005 and S013 did not develop beyond Stage 3 (first leaf present) under any of the conditions tested, while S014 seeds cultured on PhytoTechnology Orchid Seed Sowing Medium developed to Stage 4 (one leaf and ≥1 roots present; 0.5–0.6%) and 5 (two leaves and ≥1 roots present; 2.7–5.6%) under all photoperiods tested. All hybrids appeared to undergo similar early development (Stages 0–3). Limited advanced development of Hybrids S005 and S013 may be due either to inadequate culture conditions or low vigor of these hybrids. These data indicate that commercial production of some Vanda hybrids may be limited by slow growth and development. Efforts to breed hybrids which germinate and develop rapidly may be required to improve the mass market potential of Vanda hybrids.