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P Piyabhan,1 P Tingpej,2 N Duansak11Division of Physiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, KlongLuang, Pathumthani, Thailand; 2Division of Microbiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, KlongLuang, Pathumthani, ThailandBackground: Decreased gamma-aminobutyric acid (GABA)-ergic neurons in the brain of both schizophrenic patients and animal models indicates that impairment of GABAergic function is implicated in pathophysiology of the disorder. Decreased GABAergic neurotransmission might be also involved in cognitive impairment, which is developed in schizophrenia. Brahmi (Bacopa monnieri) could be a new treatment and prevention for this cognitive deficit in schizophrenia by increasing GABAergic neurons to a normal level.Aim: The authors aimed to study cognitive-enhancement- and neuroprotective-effects of Brahmi on novel object recognition memory and GABAergic neuronal density, defined by the presence of calcium binding proteins (CBPs; calbindin (CB), parvalbumin (PV), and calretinin (CR)) in a sub-chronic (2 mg/kg, Bid, ip) phencyclidine (PCP) rat model of schizophrenia.Materials and methods: In the cognitive-enhancement-effect study rats were assigned to three groups; Group-1: Control, Group-2: PCP-administration, and Group-3: PCP+Brahmi. In the neuroprotective-effect study rats were assigned to three groups; Group-1: Control, Group-2: PCP-administration, and Group-3: Brahmi+PCP. A discrimination ratio (DR) representing cognitive ability was obtained from the novel object recognition task. CB, PV, and CR immunodensity were measured in the prefrontal cortex, striatum, and cornuammonis fields 1–3 (CA1–3) using immunohistochemistry.Results: Reduced DR was found in the PCP group, which occurred alongside reduced CB, PV, and CR in all brain regions except for CR in the striatum and CA1–3 in the cognitive-enhancement-effect study. PCP+Brahmi showed a higher DR score with increased CB in the prefrontal cortex and striatum, increased PV in the prefrontal cortex and CA1–3, and increased CR in the prefrontal cortex. The Brahmi+PCP group showed higher DR score with increased CB in all areas, increased PV in the striatum, and increased CR in the prefrontal cortex and striatum.Conclusion: The present study demonstrated the effects, both partial restoration of cognitive deficit and neuroprotection, of Brahmi, and elucidated its underlying mechanism of actions via increasing GABAergic neurons in a PCP-induced schizophrenic-like model.Keywords: schizophrenia, cognitive impairment, Bacopa monnieri, calbindin, parvalbumin, calretinin

Tetrahydrocurcumin (THC) has been shown to possess anti-angiogenic activities. This study aims to investigate the effects of THC on adipose angiogenesis and expression of angiogenic factors that occurs in 60% high-fat diet-induced obese mice. Male ICR mice were randomly divided into 3 groups: mice fed with a low-fat diet (LFD group); mice fed with very high fat diet (VHFD group), and mice fed with VHFD supplemented with THC (300 mg/kg/day orally) (VHFD+THC treated group) for 6 weeks. Body weight (BW), food intake, fasting blood sugar (FBS), lipid profiles and visceral fats weight (VF) were measured. The microvascular density (MVD), TNF-α, VEGF, MMP-2, and MMP-9 expressions were evaluated. The VHFD group had significantly increased total cholesterol, triglyceride, food intake, BW, VF, VF/BW ratio, adipocyte size and the number of crown-liked structures as compared to LFD group. THC supplementation markedly reduced these parameters and adipocyte hypertrophy and inflammation in white adipose tissues. MVD, TNF-α, VEGF, MMP-2, and MMP-9 were over-expressed in the VHFD group. However, THC supplementation decreased MVD and reduced expression of TNF-α, VEGF, MMP-2, and MMP-9. In conclusion, THC suppressed angiogenesis in adipose tissue by the downregulation of TNF-α, VEGF, MMP-2, and MMP-9. With its effects on lipid metabolism as well as on food consumption, THC could contribute to lower visceral fat and body weight. Overall, our study demonstrated the potential benefit of THC in mitigating obesity and associated metabolic disorders along with elucidated the suppression of adipose angiogenesis as one of its underlying mechanisms.

Objective: To examine the effect of water extract of Thunbergia laurifolia on hepatic insulin resistance in high-fat diet-induced obese mice. Methods: High-fat diet with 45 kcal% lard fat was used for obesity induction in ICR mice. The mice were fed with high-fat diet for 16 weeks, and during the last 8 weeks, they were treated with 200 mg/ kg/day of water extracts from Thunbergia laurifolia leaf, stem and flower. Serum biochemistry, liver histology, and protein expression were examined after the treatment. Results: Extracts from all of the three parts of Thunbergia laurifolia significantly alleviated hyperglycemia, hyperlipidemia, hyperinsulinemia, and hyperleptinemia. The stem and flower extracts improved glucose tolerance. All of the extracts significantly reduced serum TNFα and monocyte chemoattractant protein-1 levels. Liver weight, triglyceride levels, and lipid accumulation were also decreased. Moreover, hepatic glucose-6-phosphatase level was significantly decreased, while the levels of PPARα, phosphorylated AMPK, and phosphorylated Akt were significantly increased with treatment of Thunbergia laurifolia extracts. Conclusions: Thunbergia laurifolia extracts can ameliorate hepatic insulin resistance in high-fat diet-induced obese mice by improving glucose and lipid homeostasis, which may be associated with stimulating phosphorylation of AMPK and Akt pathways.

Objective: To examine the effect of water extract of Thunbergia laurifolia on hepatic insulin resistance in high-fat diet-induced obese mice. Methods: High-fat diet with 45 kcal% lard fat was used for obesity induction in ICR mice. The mice were fed with high-fat diet for 16 weeks, and during the last 8 weeks, they were treated with 200 mg/ kg/day of water extracts from Thunbergia laurifolia leaf, stem and flower. Serum biochemistry, liver histology, and protein expression were examined after the treatment. Results: Extracts from all of the three parts of Thunbergia laurifolia significantly alleviated hyperglycemia, hyperlipidemia, hyperinsulinemia, and hyperleptinemia. The stem and flower extracts improved glucose tolerance. All of the extracts significantly reduced serum TNFα and monocyte chemoattractant protein-1 levels. Liver weight, triglyceride levels, and lipid accumulation were also decreased. Moreover, hepatic glucose-6-phosphatase level was significantly decreased, while the levels of PPARα, phosphorylated AMPK, and phosphorylated Akt were significantly increased with treatment of Thunbergia laurifolia extracts. Conclusions: Thunbergia laurifolia extracts can ameliorate hepatic insulin resistance in high-fat diet-induced obese mice by improving glucose and lipid homeostasis, which may be associated with stimulating phosphorylation of AMPK and Akt pathways. [ABSTRACT FROM AUTHOR]

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a significant causative agent of hospital-acquired infections. We characterized MRSA isolated from August 2012 to July 2015 from Thammasat University Hospital. Genotypic characterization of MRSA SCCmec type II and III isolates were scrutinized by whole genome sequencing (WGS). The WGS data revealed that the MRSA SCCmec type II isolates belonged to ST764 previously reported mainly in Japan. All of tested isolates contained ACME Type II′, SaPIn2, SaPIn3, seb, interrupted SA1320, and had a virulence gene profile similar to Japan MRSA ST764. Rigorous surveillance of MRSA strains is imperative in Thailand to arrest its potential spread.

Objective: To examine the effect of Brassica oleracea extract (BO) on impaired glucose and lipid homeostasis in high-fat diet (HFD)-induced obese mice. Methods: Obesity of ICR mice was induced by feeding a HFD (45 kcal% lard fat) for 16 weeks. During the last 8 weeks of study period, obese mice were additionally administered with BO (100 and 200 mg/kg/day). The metabolic parameters were determined. The gene expressions of hepatic lipogenesis were also studied. Results: After 8 weeks of treatment, BO (100 and 200 mg/kg) significantly reduced hyperglycemia and improved insulin sensitivity (P < 0.05). The serum lipid (total cholesterol, triglyceride, and non-esterified fatty acid) and hepatic triglyceride and non-esterified fatty acid were decreased (P < 0.05). The levels of insulin and leptin in serum were also decreased (P < 0.05). Moreover, the expressions of hepatic lipogenic genes including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase were decreased by BO treatment (P < 0.05). Conclusions: These results suggest that BO is a new therapeutic agent for improving the homeostasis of glucose and lipid in HFD-induced obese mice probably by suppression of lipogenic genes in liver tissue.

Objective: To evaluate the insulin sensitivity action of ferulic acid (FA) in skeletal muscle and hypothalamus of high-fat diet (HFD)-induced obese mice. Methods: Obese mouse model was induced by HFD (45 kcal% lard fat) for 16 weeks. After 8 weeks of HFD feeding, these obese mice were orally treated with FA at doses of 25 and 50 mg/kg/day for 8 weeks. At the end of all treatments, the epididymal fat, pancreas, skeletal muscle and hypothalamus were removed for biochemical parameter and protein expression examinations. Results: FA treatment significantly decreased leptin level in fat tissue and insulin level in pancreas (P < 0.05). Interestingly, obese mice treated with FA increased the protein expressions of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and phosphorylated-protein kinase B in both muscle and brain (P < 0.05). The phosphorylations of adenosine monophosphate-activated protein kinase and acetyl-CoA carboxylase in muscle, and leptin receptor protein in hypothalamus were also increased (P < 0.05). The pancreatic islets histology showed smaller size in obese mice treated with FA compared to untreated obese mice. Conclusions: These findings indicate the beneficial effect of FA in improving insulin resistance in HFD-induced obese mice. These effects are probably mediated via modulating the insulin receptor substrate/phosphatidylinositol 3-kinase/protein kinase B or adenosine monophosphate-activated protein kinase pathways.

Tetrahydrocurcumin (THC) has been shown to possess anti-angiogenic activities. This study aims to investigate the effects of THC on adipose angiogenesis and expression of angiogenic factors that occurs in 60% high-fat diet-induced obese mice. Male ICR mice were randomly divided into 3 groups: mice fed with a low-fat diet (LFD group); mice fed with very high fat diet (VHFD group), and mice fed with VHFD supplemented with THC (300 mg/kg/day orally) (VHFD+THC treated group) for 6 weeks. Body weight (BW), food intake, fasting blood sugar (FBS), lipid profiles and visceral fats weight (VF) were measured. The microvascular density (MVD), TNF-α, VEGF, MMP-2, and MMP-9 expressions were evaluated. The VHFD group had significantly increased total cholesterol, triglyceride, food intake, BW, VF, VF/BW ratio, adipocyte size and the number of crown-liked structures as compared to LFD group. THC supplementation markedly reduced these parameters and adipocyte hypertrophy and inflammation in white adipose tissues. MVD, TNF-α, VEGF, MMP-2, and MMP-9 were over-expressed in the VHFD group. However, THC supplementation decreased MVD and reduced expression of TNF-α, VEGF, MMP-2, and MMP-9. In conclusion, THC suppressed angiogenesis in adipose tissue by the downregulation of TNF-α, VEGF, MMP-2, and MMP-9. With its effects on lipid metabolism as well as on food consumption, THC could contribute to lower visceral fat and body weight. Overall, our study demonstrated the potential benefit of THC in mitigating obesity and associated metabolic disorders along with elucidated the suppression of adipose angiogenesis as one of its underlying mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yoysungnoen, Srisawat, Piyabhan, Sookprasert, Duansak, Mathuradavong, Poomipark, Munkong, Tingpej and Changtam.)

The Bacillus cereus group, also known as B. cereus sensu lato (B. cereus s.l.), is composed of various Bacillus species, some of which can cause diarrheal or emetic food poisoning. Several emerging highly heat-resistant Bacillus species have been identified, these include B. thermoamylovorans, B. sporothermodurans, and B. cytotoxicus NVH 391-98. Herein, we performed whole genome analysis of two thermotolerant Bacillus sp. isolates, Bacillus sp. B48 and Bacillus sp. B140, from an omelet with acacia leaves and fried rice, respectively. Phylogenomic analysis suggested that Bacillus sp. B48 and Bacillus sp. B140 are closely related to B. cereus and B. thuringiensis, respectively. Whole genome alignment of Bacillus sp. B48, Bacillus sp. B140, mesophilic strain B. cereus ATCC14579, and thermophilic strain B. cytotoxicus NVH 391-98 using the Mauve program revealed the presence of numerous homologous regions including genes responsible for heat shock in the dnaK gene cluster. However, the presence of a DUF4253 domain-containing protein was observed only in the genome of B. cereus ATCC14579 while the intracellular protease PfpI family was present only in the chromosome of B. cytotoxicus NVH 391-98. In addition, prophage Clp protease-like proteins were found in the genomes of both Bacillus sp. B48 and Bacillus sp. B140 but not in the genome of B. cereus ATCC14579. The genomic profiles of Bacillus sp. isolates were identified by using whole genome analysis especially those relating to heat-responsive gene clusters. The findings presented in this study lay the foundations for subsequent studies to reveal further insights into the molecular mechanisms of Bacillus species in terms of heat resistance mechanisms.

The present study investigated the activities of Raphanus sativus L. var. caudatus extract (RS) on abnormal lipid and glucose homeostasis in a high-fat diet (HFD)-induced obesity and insulin resistance in a mouse model. Institute of Cancer Research mice were rendered obese by 16-week HFD feeding. Obese mice were administered with 100 or 200 mg/kg/d RS orally during the last 8 weeks of diet feeding. Then, the biochemical parameters were determined. The gene and protein expressions regulating lipid and glucose homeostasis in the liver were measured. This study revealed that the state of hyperglycemia, hyperleptinemia, hyperinsulinemia, and hyperlipidemia was reduced after 8 weeks of RS treatment (100 or 200 mg/kg). Administration of RS also improved insulin sensitivity and increased serum adiponectin. The liver total cholesterol and triglyceride concentrations were decreased by both doses of RS. Notably, a decrease in the expression of liver-specific genes, including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase, was found in the RS-treated groups. Moreover, administration of RS showed a significant increase in the expression of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) proteins. These findings indicated that RS improved abnormal lipid and glucose homeostasis in the liver of obesity-associated insulin resistance mouse model, possibly through the stimulation of the AMPK/Sirt1 pathway., Competing Interests: AUTHOR DISCLOSURE STATEMENT The authors declare no conflict of interest., (Copyright © 2022 by The Korean Society of Food Science and Nutrition. All rights Reserved.)

Mentha cordifolia (MC) is a popular herb used to flavor food in Thailand that exhibits several biological effects. The present study aimed to determine the role of MC in regulating glucose and lipid metabolism in mice fed a high-fat diet (HFD). ICR obese mice were fed an HFD (45 kcal% lard fat) for 12 weeks, with MC (100 and 200 mg/kg/d) treatment from Week 7. After treatment with MC for 6 weeks, mice showed significantly lower rates of hyperglycemia, hyperinsulinemia, hyperleptinemia, and hyperlipidemia, and increased amounts of serum adiponectin. Furthermore, in mice treated with MC, serum interleukin-6 and tumor necrosis factor alpha were significantly inhibited and liver histology results showed decreased lipid accumulation and liver triglyceride content vs. untreated mice. In addition, MC treatment was associated with smaller fat cells and lower gene expression of liver sterol regulatory element binding protein 1c, acetyl-CoA carboxylase, and fatty acid synthase. However, MC treatment was associated with higher carnitine palmitoyltransferase 1a gene expression and significantly higher rates of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in liver, but lower levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. These results indicate MC regulates glucose and lipid metabolism in a HFD-induced obese mouse model, possibly via activation of AMPK signaling pathway., Competing Interests: AUTHOR DISCLOSURE STATEMENT The authors declare no conflict of interest., (Copyright © 2021 by The Korean Society of Food Science and Nutrition. All rights Reserved.)

We analyzed data of Staphylococcus aureus isolated from patients attending Thammasat University Hospital, Thailand from August 2012 to July 2015. In total, 232/502 (46%) S. aureus isolates were methicillin-resistant S. aureus (MRSA). There was a declining trend of proportion of MRSA infection, but the prevalence of MRSA in the last year of study remained high (38%). All 32 MRSA-infected outpatients had history of exposure to healthcare facilities during the previous two months and thus were not considered as having community-associated MRSA. In addition, all these strains were negative for pvl, suggesting that these strains were hospital-associated MRSA. All MRSA stains were susceptible to linezolid, teicoplanin and vancomycin, but resistance to erythromycin and clindamycin were nearly 100%. Fifty-two percent and 87% of MRSA strains were susceptible to tetracycline and trimethoprim-sulfamethoxazole, respectively. These results emphasize the necessity of long-term surveillance and monitoring of antimicrobial susceptibility pattern of MRSA.

Background: Ultraviolet radiation (UV) is commonly used to destroy microorganisms in the health-care environment. However, the efficacy of UV radiation against bacteria growing within biofilms has never been studied., Objective: To measure the sterilization effectiveness of UV radiation against common healthcare associated pathogens growing within biofilms., Material and Method: Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Streptococcus epidermidis, Escherichia coli, ESBL-producing E. coli, Pseudomonas aeruginosa and Acinetobacter baumannii were cultivated in the Calgary Biofilm Device. Their biofilms were placed 50 cm from the UV lamp within the Biosafety Cabinet. Viability test, crystal violet assay and a scanning electron microscope were used to evaluate the germicidal efficacy., Results: Within 5 minutes, UV radiation could kill S. aureus, MRSA, S. epidermidis, A. baumannii and ESBL-producing E. coli completely while it required 20 minutes and 30 minutes respectively to kill E. coli and P. aeruginosa. However, the amounts of biomass and the ultrastructure between UV-exposed biofilms and controls were not significantly different., Conclusion: UV radiation is effective in inactivating nosocomial pathogens grown within biofilms, but not removing biofilms and EPS. The biofilm of P. aeruginosa was the most durable.

Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various in vitro models have been developed to study P. aeruginosa pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of P. aeruginosa strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous in vitro models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of P. aeruginosa in CF.

Background and Objective: Earlier reports suggested that Pseudomonas aeruginosa frequent epidemic clones circulating in cystic fibrosis (CF) centres had increased virulence. However, recent data show no consistent associations with virulence, and suggest attenuation of virulence in chronic infection. Changes to infection control programmes in relation to frequent epidemic clones should be based on their frequency, virulence across all age groups and mode of acquisition. The Australian epidemic strain-1 (AES-1) (or the Melbourne epidemic strain) and AES-2 are common in CF clinics in mainland eastern Australia, but not in the environment. Both have shown increased virulence, but there are no data specifically in adults. This study examines the frequency and virulence of P. aeruginosa frequent epidemic clones in the adult CF clinic at Royal Prince Alfred Hospital, Sydney, Australia., Methods: Two hundred and fifty-eight P. aeruginosa isolates from 112 participants were genotyped by pulsed field gel electrophoresis. Ninety-eight patients were followed up for 1 year and associations sought between infection with a frequent epidemic clone, clinical outcome and antibiotic resistance., Results: Four frequent P. aeruginosa epidemic clones (AES-1, AES-2, S-1, S-2) affected almost 50% of participants. AES-1 predominated (38%). AES-1, AES-2 and S-1 were associated with increased exacerbations and hospital-admission days. AES-1 showed increased resistance to aminoglycosides and ticarcillin-clavulanate., Conclusions: This study supports the potential threat of frequent P. aeruginosa epidemic clones in adult CF populations.

Epidemic Pseudomonas aeruginosa have been identified in cystic fibrosis (CF) patients worldwide. The Australian Epidemic Strain-2 (AES-2) infects up to 40% of patients in three eastern Australian CF clinics. To investigate whether AES-2 isolates from chronically infected CF adults differentially express well-conserved genes potentially associated with transmissibility, we compared the transcriptomes of planktonic and biofilm-grown AES-2, infrequent P. aeruginosa clones and the reference P. aeruginosa PAO1 using the Affymetrix PAO1 array. The most interesting findings emerged from comparisons of planktonic and biofilm AES-2. AES-2 biofilms upregulated Type III secretion system genes, but downregulated quorum-sensing (QS)-regulatory genes, except lasR, QS-regulated, oxidative-stress and iron-storage genes. QS-regulated and iron-storage genes were downregulated to a greater extent in AES-2 biofilms compared with infrequent clone and PAO1 biofilms, suggesting enhanced anaerobic respiration in AES-2. Chitinase and chitin-binding protein maintained high expression in AES-2 biofilms compared with infrequent clone and PAO1 biofilms. Planktonic AES-2 upregulated QS regulators and QS-regulated genes, iron acquisition and aerobic respiration genes, and had high expression of Group III Type IV pilA compared with low expression of Group I Type IV pilA in infrequent clones. Together, these properties may enhance long-term survival of AES-2 in CF lung and contribute to its transmissibility.

Transmissible Pseudomonas aeruginosa clones potentially pose a serious threat to cystic fibrosis (CF) patients. The AES-1 clone has been found to infect up to 40 % of patients in five CF centres in eastern Australia. Studies were carried out on clonal and non-clonal (NC) isolates from chronically infected CF patients, and the reference strain PAO1, to gain insight into the properties of AES-1. The transcriptomes of AES-1 and NC isolates, and of PAO1, grown planktonically and as a 72 h biofilm were compared using PAO1 microarrays. Microarray data were validated using real-time PCR. Overall, most differentially expressed genes were downregulated. AES-1 differentially expressed bacteriophage genes, novel motility genes, and virulence and quorum-sensing-related genes, compared with both PAO1 and NC. AES-1 but not NC biofilms significantly downregulated aerobic respiration genes compared with planktonic growth, suggesting enhanced anaerobic/microaerophilic growth by AES-1. Biofilm measurement showed that AES-1 formed significantly larger and thicker biofilms than NC or PAO1 isolates. This may be related to expression of the gene PA0729, encoding a biofilm-enhancing bacteriophage, identified by PCR in all AES-1 but few NC isolates (n=42). Links with the Liverpool epidemic strain included the presence of PA0729 and the absence of the bacteriophage gene cluster PA0632-PA0639. No common markers were found with the Manchester strain. No particular differentially expressed gene in AES-1 could definitively be ascribed a role in its infectivity, thus increasing the likelihood that AES-1 infectivity is multi-factorial and possibly involves novel genes. This study extends our understanding of the transcriptomic and genetic differences between clonal and NC strains of P. aeruginosa from CF lung.

The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P = 0.02), to show elastase and alkaline protease activity by zymography (P = 0.04 and P = 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P = 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P = 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1-2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6-6.9, P<0.001]. Nine of the ten strains negative by both Western blotting and zymography were non-clonal, and all but one of these was positive for the protease IV gene. There was a marked strain-to-strain variation in the amount of protease IV produced. Secretion of protease IV by clonal strains may enhance their infectivity and ability to adapt to the changing CF lung environment. Overall the findings suggest that protease IV plays an important role in the pathogenesis of P. aeruginosa infection in the CF lung.