Pseudomonas Isolation Agar Research Articles

Bacterial Contamination of Environmental Surfaces of Veterinary Rehabilitation Clinics.

The presence of potentially pathogenic bacteria on veterinary clinic surfaces may be problematic. In this study, we collected swab samples (Fisherbrand, double transport swabs with Stuart's liquid medium) and water samples from five veterinary rehabilitation clinics. Swabs and water samples were transported to a microbiology lab for processing. At the lab, swabs were used to inoculate Hardy's Cdiff Banana Broth (for Clostridium difficile [Cdiff]) and five different types of bacterial growth media, including Hardy CHROM MRSA agar (methicillin-resistant Staphylococcus aureus [MRSA] and S. pseudintermedius [SIM]), mannitol salt agar (S. aureus [SA]), eosin methylene blue agar (enterics [ENT]), Pseudomonas isolation agar (Pseudomonas spp. [PS]), and tryptic soy agar [TSA] (non-specific). The most prominent presumptive species cultured was Cdiff (on nearly 55% of swabs). Bacillus spp. and enteric bacteria were encountered on nearly 35% of swabs, with MRSA and SIM on just over 10% of swabs. The most contaminated sample site was harnesses/life jackets used with the underwater treadmill (33% of swabs). The underwater treadmill water had total bacterial counts from 1,600 to 2,800 cfu/mL. Of all presumptive bacterial species detected, SIM tends to be more pathogenic for dogs. Targeted cleaning/disinfecting in these clinics could help reduce risks for both animals and caregivers utilizing these clinics.

Loss of AC10 Impairs Neutrophil Recruitment and Alters Bacterial Colonization in the Lung During Bacterial Pneumonia

Rationale: Pseudomonas aeruginosa is a gram-negative bacterium and a common cause of nosocomial pneumonia that can progress to acute respiratory distress syndrome (ARDS), characterized by hypoxia, pulmonary edema, elevated bicarbonate, and widespread inflammation. Neutrophil recruitment is a critical line of defense against P. aeruginosa; however, excessive, or dysregulated neutrophil infiltration can exacerbate tissue damage and contribute to the severity of ARDS. Recently, AC10 has been implicated in neutrophil transendothelial migration via CD99. Despite the growing recognition and significance of AC10 in various physiological processes, its role in neutrophil recruitment during ARDS remains poorly understood. Thus, herein we hypothesize that loss of AC10 attenuates neutrophil recruitment during pneumonia and increase bacterial colonization in the lung. Methods: Wild-type and AC10 knockout mice were intratracheally inoculated with increasing doses of P. aeruginosa. Twenty fours after infection, bronchoalveolar lavage fluid (BALF) was collected and the percent of PMNs in the BALF determined. In separate experiments, mice were intratracheally inoculated with P. aeruginosa for 6 or 24 hours and organs (lungs, kidney, liver, spleen and brain) harvested, homogenized and the homogenate cultured overnight on Pseudomonas isolation agar for quantification of colony forming units (CFUs). Results: Our data confirmed CD99 expression in rat pulmonary microvascular endothelial cells and whole lung of both wild type and AC10 knockout mice. While there was no difference in the percentage of PMNs in the BALF between male wild type and knockout animals at any of the bacterial doses tested, in the female AC10 knockout mice, there was a decrease in the percentage of cells PMNs in the BALF compared to female wild type litter mates at several doses tested (1x103 up to 1x105, p<0.05). At a higher dose of P. aeruginosa (2.5x106), there was no difference in bacterial clearance between male WT and AC10 knockout mice, but a trend toward an increase in the CFU harvested from lung tissue 6 hours post infection in female KO mice. Conclusion: Thus, CD99 is expressed in the pulmonary endothelium. Further, in female mice, AC10 plays a role in neutrophil recruitment into the airspace. Further studies are required to resolve whether AC10 plays a role in bacterial clearance in female mice in a model of P. aeruginosa pneumonia. HL121513. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Isolation and Identification of p-Nitrophenol Degrading Bacteria from Polluted Soil

Now-a-days due to an extensive industrialization, urbanization has leads concern towards organic pollution. Among the nitro-aromatic paranitrophenols are widely used as intermediates for the pesticides, dyes manufacturing processes. In the present study, a strain of Pseudomonas aeruginosa was isolated from the polluted soil especially in the vicinity of pesticide, chemical industry. This strain was able to utilize PNP as a sole source of carbon and energy by degrading it. The sub-culturing of these isolate on nutrient agar slants, growth on the Cetrimide agar formed well isolated colonies. The organism also gives bluish, greenish pigmentation on PIA (pseudomonas isolation agar). The organism is found to be gram-negative, exhibiting rods, motile. It is the catalase positive, MR positive, VP negative, Indole production is absent, it could utilize citrate as a sole source of carbon, it could rapidly liquefy gelatin and an oxidase negative. This studies have been showed that isolate NP1 have the ability to degrade the PNP. On the basis of the morphological, cultural and biochemical characteristics it was confirmed that the NP1 isolate have been identified as Pseudomonas aeruginosa.

Cupriavidus spp. and other waterborne organisms in healthcare water systems across the UK

Cupriavidus pauculus is a rare clinical pathogen, cases of which have been linked to contaminated hospital water systems. An outbreak of three cases of C.pauculus and other waterborne organisms was reported in a Glasgow hospital in 2018. To determine whether Cupriavidus spp. are present in hospital water systems elsewhere in Scotland and the UK, and to ascertain the optimal laboratory methodology for detection. This study also sought to establish where in the water system these organisms are detected, and whether a selective media could be developed for isolation. In addition, water samples were tested for the presence of other Gram-negative waterborne organisms. Water samples were received from 10 UK National Health Service hospitals and from various parts of the water system. Isolates were plated on to tryptone soya agar (TSA) and Pseudomonas isolation agar, and were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S polymerase chain reaction. Cupriavidus spp. were detected in four of 10 hospitals tested, and all five isolates were from the periphery of the water system. All hospitals had evidence of other opportunistic premise plumbing pathogens. Cupriavidus spp. were identified using TSA, with some isolates growing on Pseudomonas isolation agar; as such, they may be detected inadvertently when testing water specifically for Pseudomonas aeruginosa. Isolation of Cupriavidus spp. was not unique to the Glasgow incident, and these bacteria are present in hospital water systems elsewhere in the UK. Water testing in response to clinical cases is recommended. Consideration should also be given to water testing following bacteraemias due to other rare and unusual water-borne pathogens.

Adhesion and colonization of potential probiont Pseudomonas aeruginosa FARP72 in the intestine of yellowtail catfish, Pangasius pangasius.

Adhesion is recognized as the first important step of a probiont for intestinal colonization. This study assessed the ability of an antagonistic Pseudomonas aeruginosa FARP72 to adhere and colonize the intestine of yellowtail catfish, Pangasius pangasius both in vitro and in vivo. For the in vitro assay, the whole intestines of each of two starved P. pangasius were introduced separately into tubes containing bluish-green pigment-producing P. aeruginosa FARP72 at 8.00 log10 CFU/mL and physiological saline (0.85% sodium chloride) and incubated for 1h at 30±1°C. The homogenate mucus solutions from the intestine samples were serially diluted and plated onto Pseudomonas isolation agar to determine the counts of bluish-green pigment-producing P. aeruginosa (BPPAC). The difference between the numbers of BPPAC and presumptive Pseudomonas counts (PPC) in the treated and control intestines was attributed to the adherence of P. aeruginosa FARP72. The levels of BPPAC and PPC in the treated intestines were 6.09 ± 0.59 log10 CFU/g. Similarly, following 30days of feeding P. pangasius with P. aeruginosa FARP72 supplemented diet, the intestine of catfish recorded the BPPAC of 5.83 ± 0.64 log CFU/g. In control samples, the BPPACs were recorded as < 3.00 log10 CFU/g. The scanning electron micrograph of the intestines of P. pangasius following the in vitro and in vivo adhesion assays confirmed the ability of this bacterium to strongly adhere to the intestine, thus making it most suitable candidate probiont for use in freshwater catfish aquaculture.

High Number of Door Openings Increases the Bacterial Load of the Operating Room.

Background: Operating room (OR) traffic and door openings have emerged as potential modifiable risk factors for the development of surgical site infections. Methods: This study compared the microbial load of a Control OR without traffic versus a Simulated OR with the traffic in a typical orthopedic surgery case. Air particle counts and colony forming units (CFUs) were measured. A novel iOS app was developed to provide real-time door counts. Results: There were 1,862 particles >5.0 mcm in the Simulated OR compared with 56 in the Control OR. The CFUs from plates in the Simulated OR ranged from 4-22 (on brain heart infusion [BHI] agar), 2-266 (on mannitol salt agar [MSA]), and 1-19 (on Pseudomonas isolation agar [PIA]), while all plates in the Control OR grew 0-1 CFUs. Conclusions: High number of door openings leads to more airborne bacteria in the OR and viable bacterial on OR surfaces. The increased bacterial load throughout the OR was independent of distance from the door.

Comparison of Bacterial Contamination in a Children’s Outpatient Clinic: General Medicine Versus Pulmonary Units

Background: The bacteria that inhabit outpatient healthcare facilities influence patient outcomes and recovery, although the diversity and quantity of these bacterial communities is largely unknown. Whether differences in bacterial presence exist in individual medical specialty units of an outpatient clinic is also largely unknown. The purpose of this study was to compare bacterial species found in the general medicine and pulmonary units of an outpatient children’s clinic associated with a teaching hospital. Methods: In total, 6 locations (4 floor sites, counters, air ducts) were sampled in 3 rooms in the pulmonary (PUL) unit and 3 rooms in the general medicine (GM) unit on 13 days over a 6-month period. Sterile double transport swabs were utilized, transported on ice to a microbiology lab, and used to inoculate Hardy Diagnostics Cdiff Banana Broth (for Clostridium difficile), CHROM MRSA agar (for methicillin-resistant Staphylococcus aureus [MRSA]), eosin methylene blue (Levine-type, for Lac&amp;plus; gram negatives [GN]), and Pseudomonas isolation agar (for Pseudomonas spp and P. aeruginosa [PS and PSA]). Media were incubated for 48 hours at 37&amp;deg;C and were scored for bacterial presence based on colonial observation. Results: The presence of bacteria isolated from GM and PUL units differed by species and location. Based on the percentage of positive swabs, the presence of GN was widespread in both units (Fig&amp;nbsp;1). Additionally, bacterial presence was greatest on the floors (GN ranged from 72% to 85% on floors in the 2 units), whereas counters had fewer positive swabs (GN ranged from 23% to 38% on counters), and swabs from return air ducts rarely led to bacterial growth. The 1 case in which swabs from the PUL unit resulted in higher levels of bacterial growth than for the GM unit was for PSA (GM, 8%; PUL, 13%). C. difficile detection was the same on both units (ie, 35% of floor samples showed contamination). Conclusions: The levels of environmental bacterial presence observed for these clinic units differed in some cases by unit and ranged from not detectable to very high levels. Detection of C. difficile on 35% of floor samples in both units could be problematic. Additionally, for the PUL unit, contamination of 13% of floor samples by PSA should raise concerns because many patients in this clinic have cystic fibrosis (CF). Although many CF patients are colonized by PSA, others may potentially contract an infection by this pathogen from the clinical environment. This observation supports current infection control recommendations for CF patients in outpatient settings.Funding: NoneDisclosures: None

Bacterial Colonization of Waiting Rooms in a Newly Constructed Children’s Outpatient Clinic: Construction Through 6 Months After Opening In Waiting Rooms

Background: Healthcare-associated infections (HAIs) represent an ongoing problem for all clinics. Children’s clinics have waiting rooms that include toys and activities to entertain children, possibly representing reservoirs for HAIs. This study focuses on a newly constructed children’s outpatient clinic associated with a teaching hospital. We studied waiting room bacterial colonization of floors and play devices from the last phase of construction through 6 months of clinical use. Methods: Waiting room areas on the first 2 floors of the facility were studied due to high patient volume in those areas. In total, 16 locations were sampled: 11 on floors and 5 on play items. Using sterile double-transport swabs, all locations were sampled on 5 separate occasions over 2 months during the last phase of construction and 13 times over 6 months after the clinic was opened. After collection swabs were placed on ice, transported to a microbiology lab, and used to inoculate Hardy Diagnostics Cdiff Banana Broth (for Clostridium difficile - Cdiff), CHROM MRSA agar (for methicillin resistant Staphylococcus aureus - MRSA), Pseudomonas isolation agar (for Pseudomonas spp and P. aeruginosa), and tryptic soy agar to detect Bacillus spp. Media were incubated for 48 hours at 37°C and were scored for bacterial presence based on observation of colonies or change in the medium. Results: During the construction phase, waiting-room-floor bacterial colonies were dominated by Bacillus spp, and first-floor waiting rooms had nearly 7 times more colonies than those on the second floor (P &lt; .05). A similar pattern was observed for C. difficile and MRSA. No Pseudomonas spp were observed during construction. Once patients were present, Bacillus spp contamination dropped for the first floor, but increased for the second floor. All other bacterial types (C. difficile, MRSA, Pseudomonas spp, and P. aeruginosa) increased on the second floor after the clinic opened (eg, from 23% to 42% for C. difficile and from 7% to 46% for MRSA; P &lt; .05). The play devices showed small increases in bacterial load after clinic opening, most notably Pseudomonas spp. Conclusions: This study provides evidence that a shift from bacterial species associated with soil (eg, Bacillus spp) toward species commonly associated with humans occurred in waiting rooms after construction in this children’s outpatient clinic. Increases for MRSA, Pseudomonas spp, and P. aeruginosa were linked to patient presence. These data suggest that patients, their families, and clinic staff transport bacteria into clinic waiting rooms. This outpatient clinic environmental contamination may increase potential for HAIs and may represent a target for intervention.Funding: NoneDisclosures: None

Characterization of Highly Antibiotic Resistant Bacteria found in an Urban Waterway

Pseudomonas are a group Gram‐negative Gammaproteobacteria with numerous different species that are a risk of causing bacterial infections. The goal is to measure how many Pseudomonas are present in different urban waterways, to evaluate if they are resistant to antibiotics and the degree of resistance they show, and to determine the mechanisms of resistance that they utilize. The research was conducted on urban waterways in the suburban Chicago region. Pseudomonas Isolation Agar (PIA) is a selective media that enriches for Pseudomonas. Water from the urban waterways was grown on PIA plates containing antibiotics, which allowed the level of resistance to be measured and evaluated based on qualitative growth. An antibiotic panel was chosen that provides a broad set of antibiotics which will each interact with the bacteria differently. Growth assays at varying concentrations of antibiotic allow for the degree of resistance to be evaluated. Whole genome sequencing of highly antibiotic resistant Pseudomonas allows for a bigger picture look at which genes make the bacteria resistant. We have found massive resistance in the waterways in several of the antibiotics. The hope of this work is to characterize potentially dangerous microbes that live in urban waterways.

Isolation and characterization of nitrogen removal bacteria from sewage and fecal sludge of dairy cow farms in Cu Chi, Ho Chi Minh City, Vietnam

The bacteria that involved in the removal of nitrogen in wastewater were initially studied. Isolation media were SW-MM (artificial Seawater-Minimal Medium) and 17208 PIA (Pseudomonas Isolation Agar). The pure isolates were streaked on the SW-MM agar plates that added ammonia, nitrate, or nitrite with ascending concentrations to select the isolates had good nitrogen removal potential. The result was that 49 isolates had been collected from 11 samples of sewage and fecal sludge of dairy cow farms, in which including 6 isolates had been isolated by using specialized medium for Pseudomonas. All 49 isolates (100%) were able to grow on the SW-MM added 100 mM ammonia/nitrate or more. The ability to withstand ammonia and nitrate of the best isolates reaches 800 mM. Meanwhile, the ability to withstand nitrite had only belonged to 39 isolates (79.6%) with nitrite concentrations of 5 - 15 mM. Nine isolates were investigated the in vitro ability to reduce ammonia in the wastewater. Quantitative results of ammonia by phenol-hypochlorite method showed that the ability to reduce NH4+ of 9 isolates ranged from 27.9 – 48.9 mg/L on 10 days after inoculation. The six best isolates that had the ammonia- removing efficiency reached 88.5% - 97.5% were identified by using the MALDI Biotyper System (Germany). The identification result showed that XM3, NA6 was Bacillus megaterium, M7C1 was Bacillus pumilus, NA8 was Rhodococcus ruber, MD6 was Pseudomonas fulva, and XM5 was Pseudomonas mendocina. It is recommended to test the ability of these isolates to reduce nitrate and nitrite in the wastewater.

Deciphering rhizosphere microbiome for the development of novel bacterial consortium and its evaluation for salt stress management in solanaceous crops in India

Eighty bacteria were isolated from the salt affected solanaceous crops (potato, tomato, chilli and brinjal) rhizosphere and fallow land in Mau, Faizabad, Ballia, Gazipur and Varanasi districts of Uttar Pradesh, India. All the bacterial isolates showed morphological variation on Nutrient agar, Jenson’s medium, Modified Czapek-Dox medium, Mannitol egg yolk polymyxin agar, Pseudomonas isolation agar medium, Luria Bertni agar, Tryptic soya agar, King B agar and Nutrient broth yeast extract. Majority of the isolates showed creamy colony (32.5%), spherical (66.25%), shiny (88.7%), raised (62.25%), translucent (71.25%) and entire type of surface margin (78.75%). The gram staining, catalase test, glucose fermentation, IAA test, H2S production, starch hydrolysis, tween 80, gelatin hydrolysis, casein hydrolysis, P solubilization, Zn solubilization and ammonia production tests revealed that 26, 28, 13, 3, 11, 10, 18, 10, 17, 24, 23 and 16 isolates were positive for these tests respectively. The molecular characterization of bacterial isolates Brevibacillus fluminis, Brevibacillus agri, Bacillus paralicheniformis, and Microbacterium arborescens revealed that these are highly salt tolerant (18%) produced 37 alleles using box primer. The number of bands (400–3190 bp) varied from 1 to 10. A cluster analysis with unweighted pair-group method with arithmetic means (UPGMA) based on Jaccard’s similarity coefficient divided the bacterial isolates into two major and three minor clusters with coefficient of similarities ranging from 1 to 23%. Phylogenetic analysis of salt tolerant bacterial isolates showed that out of 22 isolates, 17 (77.27%) were found to be phylogenetically closely related to Bacillus, with 99–100% similarity in their 16S rDNA sequences, making Bacillus the most dominant genus among salt tolerant bacteria in the study.

Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination.

A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspensions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin methylene blue and MacConkey detected only E. coli RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the background fluorescence was referred to as quantification cycle. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. Based upon standard curve analysis of B. cepacia DNA, the minimum DNA concentration that could be detected was 10 fg/uL with a correlation value of 0.98. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.LAY ABSTRACT: A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. B. cepacia is the number one reason for microbial contamination recalls of non-sterile drug products in the USA. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.

Hospital microbial surface colonization revealed during monitoring of Klebsiella spp., Pseudomonas aeruginosa, and non-tuberculous mycobacteria

Hospital environmental conditions, human occupancy, and the characteristics of the equipment influence the survival of microbial communities and raise a concern with regard to nosocomial infections. The objective of the present work was to use the monitoring of Pseudomonas aeruginosa, Klebsiella spp. and non-tuberculous mycobacteria as a strategy to improve knowledge on microbial colonization of non-critical equipment and surfaces, in a tertiary hospital from Central Portugal. A 3-month microbiological survey was performed in a district teaching hospital. A total of 173 samples were obtained from the wards Hematology, Urology, Medicine, and Renal Transplants, and 102 presumptive strains recovered. Per sampling, Pseudomonas Isolation agar showed 42.8 to 73.3% of presumptive P. aeruginosa colonies and MacConkey agar recovered mostly Staphylococcus. Most of the colonies recovered in Middlebrook 7H10-PANTA belonged to the genus Methylobacterium. Taps and WC shower curtains carry high bacterial species diversity. The Redundancy Analysis grouped the samples in those mostly handled by patients, and those mostly handled by healthcare staff or of mixed use. This study shows that the preferential users of the space and equipment seem to be important contributors to the microbial community. The most recovered genus was Methylobacterium, known as colonizer of the water distribution system therefore, it is possible that the water points and biofilms in taps also contribute as dispersion hotspots.

Molecular-based analysis of the bacterial community structure of spoilt latex paints in Nigeria

Biodeteriorated paints showing discolouration, loss of viscosity and foul odour have resulted in substantial economic losses in Nigeria. The present study focused on enumeration and identification of bacteria isolated from spoilt paints made in Nigeria using a non-selective nutrient agar (NA) and a semi-selective Pseudomonas Isolation Agar (PIA). The bacterial population density in a fresh paint sample was 3.0 x 103 CFU/ml while that of five samples of spoilt paint ranged from 1.0 x 103 to 1.2 x 106 CFU/ml on NA. The fresh paint had no growth on PIA while spoilt paint bacterial counts ranged from 2.0 x 103 to 8.0 x 105 CFU/ml. The most diverse colony types from the spoilt paint samples were randomly selected for purification. We subjected 9 isolates to sequencing of a portion of the 16S rRNA gene and 14 isolates to whole-cell fatty acid methyl ester profiling using the MIDI-FAME system. The most prevalently identified were Klebsiela and Providencia species. A subset of isolates were examined by transmission electron microscopy. Other detected genera included Bacillus, Brevibacillus, Lysinibacillus, and the Nocardioforms. Our results show that spoilt latex paints made in Nigeria can harbour diverse bacterial species which can cause biodeterioration.

Glutamine supplementation in cystic fibrosis: A randomized placebo-controlled trial.

Pulmonary infection and malnutrition in cystic fibrosis are associated with decreased survival. Glutamine has a possible anti-microbial effect, with a specific impact against Pseudomonas aeruginosa. We aimed to test the hypothesis that oral glutamine supplementation (21 g/day) for 8 weeks in adults with cystic fibrosis would decrease pulmonary inflammation and improve clinical status. The study design was a randomized double-blind placebo-controlled study design with an iso-nitrogenous placebo. The primary analysis was intention to treat, and the primary outcome was change in induced sputum neutrophils. Thirty-nine individuals were recruited and thirty-six completed the study. Glutamine supplementation had no impact on any of the outcome measures in the intention-to-treat analysis. In the per protocol analysis, glutamine supplementation was associated with an increase in induced sputum neutrophils (P = 0.046), total cells (P = 0.03), and in Pseudomonas isolation agar colony forming units (P = 0.04) compared to placebo. There was no effect of glutamine supplementation on markers of pulmonary inflammation in the intention-to-treat analysis.

Application of lavender and rosemary essential oils improvement of the microbiological quality of chicken quarters

The aim of the present work was monitoring of chicken quarters microbiological indicators after treatment by ethylenediaminetetraacetate (EDTA), lavender (Lavandula angustifolia L.) and rosemary (Rosmarinus officinalis L.) essential oil, stored under vacuum packaging, at 4 ±0.5°C for a period of 16 days. The following treatments of chicken quarters were used: Air-packaging control samples, control vacuum-packaging samples, vacuum-packaging with EDTA solution 1.50% w/w, control samples, vacuum-packaging with Lavandula angustifolia essential oil at concentrations 0.2% v/w and vacuum-packaging with Rosmarinus officinalis essential oil at concentration 0.2% v/w. The quality assessment of all samples was established by microbiological analysis. Sampling was carried out after certain time intervals: 0, 4, 8, 12 and 16 days. Chicken quarters were stored under vacuum packaging, at 4 ±0.5°C during experiment. Microbiological analyses were conducted by using standard microbiological methods. Anaerobic plate count were determined using Plate Count Agar, after incubation for 2 days at 35°C under anaerobic condition. Pseudomonas spp. were determined on Pseudomonas Isolation agar after incubation at 48 h at 25°C. For lactic acid bacteria were inoculated into Rogosa and Sharpe agar after incubation 48-78 h at 37°C in an aerobic atmosphere supplemented with carbon dioxide (5% CO2). For members of the family Enterobacteriaceae violet red bile glucose agar were used and samples were incubated at 37°C for 24 h. The initial APC value of chicken quarter was 3.00 log CFU.g-1 on 0 day. The number of anaerobic plate count ranged from 3.00 log CFU.g-1 in all tested group on 0 day to 6.11 log CFU.g-1 on 16 day in control group stored in air condition. The initial LAC value of chicken quarter was 3.00 log CFU.g-1 on 0 day. The number of lactic acid bacteria ranged from 3.00 log CFU.g-1 in all tested group on 0 day to 3.58 log CFU.g-1 on 16 day in control group stored in air condition. The initial Enterobacteriacea genera value of chicken quarter was 2.00 log CFU.g-1 on 0 day. Presences of these bacteria were found on all groups at 16 days. The results of this present study suggest the possibility of application the Lavandula angustifolia and Rosmarinus officinalis essential oil as natural food preservatives and potential sources of antimicrobial ingredients for food industry.

WS19.5 Alginate phenotypes of Pseudomonas aeruginosa from a Brazilian CF center compared to Scandinavian CF centers

Objectives Alginate production (mucoid phenotype) of P. aeruginosa is a marker of chronic biofilm infection in the lungs of CF patients. Little is known about the occurrence of alginate-producing P. aeruginosa in CF patients outside Europe and North America. We therefore decided to study the occurrence of the alginate phenotype in P. aeruginosa from chronically infected CF patients in a CF center in Brazil and CF centers in Scandinavia (Ciofu et al., 2008). Methods The phenotype was classified according to Schurr et al. (1994): Type I: mucoid (M) on PIA ( Pseudomonas isolation agar) and LB agar, type II: mucoid on PIA but non-mucoid (NM) on LB, type III: non-mucoid on both, type IV: very slight but detectable mucoidy on both media after ≥4 days incubation. Results See the table. TableDistribution of different alginate phenotypesType I TypeII Type III Type IVBoth M&NM isolates from 20 Brazilian patients95%40%65%Both M&NM isolates from 83 Scandinavian patients89%17%87%6%Only NM isolates from 12 Brazilian patients92%8%Only NM isolates from 8 Scandinavian patients75%25% Conclusion Generally, the results show rather similar distributions of the Brazilian and Scandinavian isolates on the different phenotypes (Table). This probably reflects common selective environment in the lungs of CF patients in Brazil and Scandinavia, since the countries are widely separated and have completely different climate and geography, whereas the public medical systems are tax-based in all 4 countries. We are now sequencing the mucA and algT genes of the isolates which are responsible for the alginate phenotypes.

CHARACTERIZATION OF ENDOPHYTIC MICROFLORA OF ROSA CANINA FRUITS

There aren’t a lot of studies about the bacterial communities associated with the Rosa canina and the aim of this study was to characterize endophytic bacteria from fruit of Rosa canina. The fruits of R. canina, which is growing wild in Slovakia, were collected in May 2013 from four locations: Nitra-Zobor, Vrbové-Baraní dvor, Rišňovce, Modra pažiť, Slovakia. Microbiological analyses were conducted by use of standard microbiological methods by spreading of fruits homogenates onto agar plate. Total viable count and mesophilic anaerobic sporulating bacteria were determined on Plate Count Agar after incubation for 2 days at 37 °C. Pseudomonas aeruginosa enumeration was carried out after incubation of Pseudomonas Isolation agar at 48 h at 35 °C. For members of the family Enterobacteriaceae (45 °C) Violet Red Bile Glucose agar were used and incubation was carried out for 24 h at 37 °C. For determinations of fungal colonies Malt agar and Czapek-Dox agar were inoculated using the spread-plate technique and incubated at 25 °C for 5 days. The yeasts were grown in Glucose Yeast Peptone agar (aerobiosis) at 25 °C during 72 hours. The total viable count of fruits ranged from 4.07 log cfu.g-1 in Rišňovce to 4.84 log cfu.g-1 in Vrbové Baraní dvor. Number of mesophilic anaerobic sporulating bacteria ranged from 4.09 in Vrbové Baraní dvor to 4.82 log cfu.g-1 in Modrá pažiť. Number of Pseudomonas aeruginosa count ranged from 2.00 in Nitra Zobor and Vrbové Baraní dvor to 3.94 log cfu.g-1 in Modrá pažiť. In our study the number of Enterobacteriaceae genera ranged from 3.38 in Nitra Zobor to 4.25 log cfu.g-1 in Vrbové Baraní dvor. Number of yeasts ranged from 3.36 in Vrbové Baraní dvor to 3.85 log cfu.g-1 in Modrá pažiť. Number of microscopic filamentous fungi ranged from 2.60 in Modrá Pažiť to 3.52 log cfu.g-1 in Nitra Zobor. Our findings indicate that Rose plant is naturally associated with a variety of endophytic microorganisms, which have different physiological and biochemical properties.

Identification of Novel Genes Associated with Alginate Production in &lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt; Using Mini-&lt;em&gt;himar1&lt;/em&gt; Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ(22)). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Evaluation of five selective media for the detection of Pseudomonas aeruginosa using a strain panel from clinical, environmental and industrial sources

Isolation and correct identification of the opportunistic pathogen and industrial contaminant Pseudomonas aeruginosa are very important and numerous selective media are available for this purpose. A novel comparison of five selective media having positive (acetamide-based agars), negative (Pseudomonas CN selective agar [Oxoid Ltd.] and Pseudomonas Isolation agar [Sigma-Aldrich Company Ltd.]) and chromogenic (chromID® P. aeruginosa [bioMérieux]) selection strategies was performed using a systematically designed bacterial test panel (58 P. aeruginosa and 90 non-P. aeruginosa strains including those commonly misidentified as P. aeruginosa by culture-dependent techniques). Standardised inocula were added to the selective media and the results were recorded after 24 and 72h. After 72h of incubation at 37°C chromID® P. aeruginosa displayed the highest specificity (70%) and had good sensitivity (95%), although the sensitivity was negatively impacted by the large variation in colour of P. aeruginosa colonies, which hampered interpretation. Both media containing inhibitory selective agents performed very similarly, both having 100% sensitivity and a specificity of approximately 30%. Raising the incubation temperature to 42°C increased the specificity of Pseudomonas CN selective agar and Pseudomonas isolation agar (61% and 47% respectively after 72h), but increased the number of false positives encountered with the chromogenic medium, decreasing its specificity to 68% after 72h. Growth on the acetamide agars was weak for all strains and it was often difficult to determine whether true growth had occurred. This, compounded by the low specificity of the acetamide agars (<26%), suggested they were less suitable for application to clinical or industrial settings without further modification. Overall, the chromogenic agar was the most selective but further consideration is required to optimise interpretation of results.