Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as an Anti-biofilm Agent Against Non-Candida albicans Candida Species.

BACKGROUND: The predominance of non-Candida albicans Candida (NCAC) species causing healthcare-associated infections has increased over the last decade pertaining to their ability to form biofilms on medical devices. These biofilm-associated infections are challenging to treat as they are resistant to antifungal agents and evade host-immune response resulting in a high risk of device failure or biomaterial removal. Thus, to minimize the risk of biofilm-associated infections, preventing biofilm formation is the best approach which is mediated by the quorum quenching process. METHODS: The present study investigated the modulatory effect of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) on NCAC biofilm formation and also assessed the effect of the DMHF-coated catheters on biofilm formation of NCAC. The NCAC isolates studied were Candida tropicalis, Candida glabrata and Candida krusei isolated from catheter tip, urine and blood, respectively. RESULTS: DMHF at a concentration of 30 µg/mL showed an inhibitory effect against NCAC biofilms at various stages and was statistically significant (p ≤ 0.05) against the various concentrations (50-5 µg/mL) tested and also among the three phases of experiment. The furanone content on coated catheters ranged from 170 to 750 µg and release of furanone from the coated catheter was about 15 µg for 30 days. The effect of DMHF-coated catheters on NCAC biofilm formation was observed by the scanning electron microscopy which revealed the absence of NCAC adherence on DMHF-coated catheters. DISCUSSION: This study provides a design to develop furanone-coated biomaterials which could be implemented in healthcare settings to reduce medical device-associated infections. The excellent biological performance, combined with their antimicrobial properties, suggests that 2,5-dimethyl-4-hydroxy-3(2H)-furanone could be an effective anti-infective coating for implantable devices.

Emerging Pathogens in Planetary Health and Lessons from Comparative Genome Analyses of Three Clostridia Species.

Clostridioides difficile (CD) is a major planetary health burden. A Gram-positive opportunistic pathogen, CD, colonizes the large intestine and is implicated in sepsis, pseudomembranous colitis, and colorectal cancer. C. difficile infection typically following antibiotic exposure results in dysbiosis of the gut microbiome, and is one of the leading causes of diarrhea in the elderly population. While several studies have focused on the toxigenic strains of CD, gut commensals such as Clostridium butyricum (CB) and Clostridium tertium (CT) could harbor toxin/virulence genes, and thus pose a threat to human health. In this study, we sequenced and characterized three isolates, namely, CT (MALS001), CB (MALS002), and CD (MALS003) for their antimicrobial, cytotoxic, antiproliferative, genomic, and proteomic profiles. Although in vitro cytotoxic and antiproliferative potential were observed predominantly in CD MALS003, genome analysis revealed pathogenic potential of CB MALS002 and CT MALS001. Pangenome analysis revealed the presence of several accessory genes typically involved in fitness, virulence, and resistance characteristics in the core genomes of sequenced strains. The presence of an array of virulence and antimicrobial resistance genes in CB MALS002 and CT MALS001 suggests their potential role as emerging pathogens with significant impact on planetary health.

Dysbiosis of the Beneficial Gut Bacteria in Patients with Parkinson's Disease from India.

Objectives: Recent advancement in understanding neurological disorders has revealed the involvement of dysbiosis of the gut microbiota in the pathophysiology of Parkinson's disease (PD). We sequenced microbial DNA using fecal samples collected from PD cases and healthy controls (HCs) to evaluate the role of gut microbiota. Methods: Full-length bacterial 16S rRNA gene sequencing of fecal samples was performed using amplified polymerase chain reaction (PCR) products on the GridION Nanopore sequencer. Sequenced data were analyzed using web-based tools BugSeq and MicrobiomeAnalyst. Results: We found that certain bacterial families like Clostridia UCG 014, Cristensenellaceae, and Oscillospiraceae are higher in abundance, and Lachinospiracea, Coriobacteriaceae and genera associated with short-chain fatty acid production, Faecalibacterium, Fusicatenibacter, Roseburia and Blautia, are lower in abundance among PD cases when compared with the HC. Genus Akkermansia, Dialister, Bacteroides, and Lachnospiraceae NK4A136 group positively correlated with constipation in PD. Conclusion: Observations from this study support the other global research on the PD gut microbiome background and provide fresh insight into the gut microbial composition of PD patients from a south Indian population. We report a higher abundance of Clostridia UCG 014 group, previously not linked to PD.

Following the Indian Immigrant: adoption of westernization results in a western gut microbiome and an increased risk of inflammatory bowel diseases.

Indians who migrate to westernized countries such as Canada, the USA, and the UK are at an increased risk of developing inflammatory bowel disease (IBD). While the underlying aetiology of IBD remains unclear, a gut microbiome, i.e. no longer symbiotic with its host, is a major player. Increasing IBD incidence in Indian immigrants may be due to the adoption of western practices that result in loss of tolerance of a symbiotic community in the gut and its underlying immune responses. However, little is known about the microbial changes in the Indian gut, including shifts in the microbiome when they migrate to westernized countries. In this Current Opinion, we discuss what is known about the Indian gut microbiome and how living in a westernized environment may be impeding what was once a symbiotic relationship with their gut microbiome and intestinal mucosae, which may be the driving factor in their increased risk of IBD.

Exploring the multifactorial aspects of Gut Microbiome in Parkinson's Disease.

Advanced research in health science has broadened our view in approaching and understanding the pathophysiology of diseases and has also revolutionised diagnosis and treatment. Ever since the establishment of Braak's hypothesis in the propagation of alpha-synuclein from the distant olfactory and enteric nervous system towards the brain in Parkinson's Disease (PD), studies have explored and revealed the involvement of altered gut microbiota in PD. This review recapitulates the gut microbiome associated with PD severity, duration, motor and non-motor symptoms, and antiparkinsonian treatment from recent literature. Gut microbial signatures in PD are potential predictors of the disease and are speculated to be used in early diagnosis and treatment. In brief, the review also emphasises on implications of the prebiotic, probiotic, faecal microbiota transplantation, and dietary interventions as alternative treatments in modulating the disease symptoms in PD.

Microbiome Profiling of Enterotoxigenic Escherichia coli (ETEC) Carriers Highlights Signature Differences between Symptomatic and Asymptomatic Individuals.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children in low- and middle-income countries (LMICs). However, large-scale pathogen burden studies in children have identified ETEC in the guts of both symptomatic patients and controls. The factors that influence this balance are poorly understood, but it is postulated that the gut microbiome may play a role in either resistance or progression to disease. In this study, we profiled the microbiomes of children and adults from Bangladesh who were asymptomatically or symptomatically infected with ETEC. Symptomatic patients had significantly higher numbers of sequenced reads mapping to both E. coli and two ETEC toxins, suggesting higher bacterial burden. They were also significantly more likely to be coinfected with enteroaggregative E. coli (EAEC) and had higher proportions of other Gammaproteobacteria, including Klebsiella, Salmonella, and Haemophilus. Colonization with ETEC was also associated with increased prevalence of antimicrobial resistance (AMR) genes, most notably those of the ß-lactamase class. Taxonomic profiles were distinctly different between all groups in both species richness and composition, although the direction of these changes was different in adults and children. As seen previously, children with high E. coli burdens also had higher proportions of Streptococcus spp., while healthy children were more heavily colonized by Bifidobacterium spp. Our study provides insight into the microbiome changes that occur upon infection with ETEC in an endemic setting and provides rationale for future studies investigating how the microbiome may protect or predispose individuals to symptomatic infections with gastrointestinal pathogens. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children in low- and middle-income countries. However, these bacteria are often identified in both patients and healthy controls. We do not yet understand why only some people get sick, but it has been suggested that the gut microbiome might play a role. In this study, we used metagenomic sequencing to profile the gut microbiomes of individuals in Bangladesh, with or without a symptomatic ETEC infection. In general, individuals with high levels of ETEC also harbored other pathogenic E. coli strains, higher proportions of Gammaproteobacteria such as Salmonella and Klebsiella, and a higher burden of antimicrobial resistance genes in their guts. Healthy children, in contrast, had higher levels of bifidobacteria. These data confirm that the composition of the gut microbiome is different between symptomatic and asymptomatic people and provides important preliminary information on the impact of the gut microbiome in intestinal infections.

Mono- and Bimetallic Nanoparticles for Catalytic Degradation of Hazardous Organic Dyes and Antibacterial Applications.

In the present work, gold (Au), silver (Ag), and copper (Cu) based mono- and bimetallic NPs are prepared using a cost-effective facile wet chemical route. The pH for the synthesis is optimized in accordance with the optical spectra and supported by the finite difference time domain simulation studies. FESEM and TEM micrographs are used to analyze the morphology of the prepared nanoparticles. TEM images of bimetallic nanoparticles (BMPs) verified their bimetallic nature. XRD studies confirmed the formation of fcc-structured mono- and bimetallic NPs. Photoluminescence studies of the as-synthesized NPs are in good agreement with the previous publications. These synthesized NPs showed enhanced catalytic activity for the reduction/degradation of 4-nitrophenol, rhodamine B, and indigo carmine dyes in the presence of sodium borohydride (NaBH4) compared to NaBH4 alone. For the reduction of 4-nitrophenol, Au, Cu, and CuAg nanoparticles exhibited good catalytic efficiency compared to others, whereas for the degradation of rhodamine B and indigo carmine dyes the catalytic efficiency is comparatively high for CuAg BMPs. Furthermore, the antibacterial assay is carried out, and Ag NPs display effective antibacterial activity against Klebsiella pneumoniae, Salmonella ser. Typhimurium, Acinetobacter baumannii, Shigella flexneri, and Pseudomonas aeruginosa.

Profile of Helicobacter pylori cagA &vacA genotypes and its association with the spectrum of gastroduodenal disease.

PURPOSE: Globally, H. pylori virulence factors cagA and vacA genotypes and its variation is leading to the austere form of the gastroduodenal disease. Our objectives were to detect H. pylori in dyspeptic patients from biopsy samples with the validation of the various existing diagnostic tools and to screen the cagA, vacA genotypes profile from biopsy specimens and how it impacts in progression of gastroduodenal disease in southern India. METHODS: 374 patients who attended endoscopy unit at Kasturba Hospital, Manipal with their consent obtained their biopsies. H. pylori were detected by HPE, Culture, RUT and PCR and its virulence gene were patterned with PCR. RESULTS: The positive rate of H. pylori by HPE, RUT, Culture and PCR were 51.33%, 47.1%, 32.4% and 50.3% respectively and comparison by Bayesian LCMs analysis showed PCR is superior among them. The frequency of H. pylori virulence gene viz cagPAI (cagA) were 80.9%, and vacA alleles-s1m1 (42%), s1m2 (33%) and s2m2 (25%) genotypes by PCR respectively. Four combinations of cagA/vacA genotypes were noted, majority of strains harboured cagA+/vacA s1m1 genotypes (42.6%), interestingly this hyper-virulent strain more frequently seen in severe gastroduodenal disease whereas cagPAI negative strains as well as cagA-/vacA s2m2 combinations (19.1%) are seen most commonly in functional dyspepsia cases and depicted significant association by Chi-square test. CONCLUSIONS: This study validates and compares the existing diagnostic methods for detecting H. pylori in biopsies. Also, it reveals some pattern of virulence gene combination will play a vital role in disease progression.

Is the antiseizure effect of ketogenic diet in children with drug-resistant epilepsy mediated through proinflammatory cytokines?

In order to understand whether the antiseizure mechanism of ketogenic diet (KD) is mediated through its anti-inflammatory effect, we measured the serum concentrations of cytokines IL- 1ß and IL-6 in 21 children with drug-resistant epilepsy. We found a significant reduction in the levels of serum IL- 1ß and IL-6 levels at one-year of KD therapy compared to baseline. However, we did not find any correlation between decrease in the serum concentrations of these interleukins with the reduction in seizure frequency at one-year of KD therapy, which may be due to the small sample size and heterogeneous patient population we studied. Future studies should try to overcome these limitations.

Thymol, a naturally occurring monocyclic dietary phenolic compound protects Chinese hamster lung fibroblasts from radiation-induced cytotoxicity.

The effect of thymol (TOH), a dietary compound was investigated for its ability to protect against radiation-induced cytotoxicity in Chinese hamster lung fibroblast (V79) cells growing in vitro. Treatment of V79 cells with 25 microg/ml of TOH prior to 10 Gy gamma radiation resulted increase in the cell viability than that of radiation alone as evaluated by MTT assay. Similarly, there was a significant increase in the surviving fraction observed with 25 microg/ml of TOH administered 1h prior to graded doses of gamma radiation. Further, 25 microg/ml TOH treatment before irradiation significantly decreased the percentage of radiation-induced apoptotic cells (sub-G(1) population) analyzed by flow cytometry as well as DNA ladder assay. TOH was found to inhibit various free radicals generated in vitro, viz., DPPH, O(2), ABTS(+) and OH in a concentration dependent manner. TOH also inhibited the radiation-induced decrease in intracellular glutathione, superoxide dismutase and catalase enzyme levels in V79 cells accompanied by the reduction in lipid peroxides. Our study demonstrated antagonistic potential of TOH against radiation-induced oxidative stress, lipid peroxidation resulting in increased cell viability.

Poultry: a receptacle for non-typhoidal Salmonellae and antimicrobial resistance.

BACKGROUND AND OBJECTIVES: Non-typhoidal Salmonellosis, a zoonotic infection associated with acute gastroenteritis is caused by non-typhoidal salmonellae (NTS). The study was carried out to determine the prevalence of NTS serovars and their antimicrobial resistance along with the presence of the virulence gene (invA gene) in poultry samples. MATERIALS AND METHODS: This is a prospective cross-sectional study carried out at the Enteric Diseases Division, Kasturba Medical College, Manipal, South India from January 2016- December 2017. Poultry samples were collected randomly from two local poultry farms in Udupi district and processed following CDC standard protocol. RESULTS: From the 396 poultry meat samples, intestinal contents and faecal samples collected, 58 NTS serovars were isolated showing a prevalence of 14.64%. Salmonella Infantis, 43.1%, 25/58 was the commonest serovar. Resistance to ciprofloxacin 72.41%, ampicillin 32.8%, gentamicin 17.24%, cotrimoxazole 29.31% and amoxicillin-clavulanic acid 6.9% was observed. The invA gene was detected in 43 NTS isolates (74.13%). CONCLUSION: Poultry sources are recognized as a significant cause for non-typhoidal salmonellosis. Therefore, hygienic measures should be initiated to reduce the contamination of meat and poultry products with virulent strains of Salmonella that are of public health significance.

Oral candidal carriage correlates with CD4+ cell count but not with HIV and highly active antiretroviral therapy status.

AIM: The occurrence of oropharyngeal candidiasis (OPC) may be influenced by oral candidal carriage (OCC). Although OPC is strongly associated with low CD4+ cell count (400-700 cells/mm3 ) and a lack of highly active antiretroviral therapy (HAART), the effect of these two parameters on OCC is debatable. We investigated the oral candidal carriage, species diversity, antifungal susceptibility and the association of OCC with CD4+ cell count and HAART. METHODS: Oral candidal isolates from 120 HIV+ patients (60 receiving and 60 not receiving HAART) and 60 healthy controls were quantified, and their species determined using standard culture and biochemical methods, followed by antifungal susceptibility testing using the agar dilution method. RESULTS: The OCC was significantly higher in HIV+ patients; Candida albicans was the most frequently isolated species in both groups, followed by Candida tropicalis. Candidal density carriage correlated significantly with CD4+ cell count, but not with HIV and HAART status. Among the isolates from HIV+ patients, 35.4% showed reduced susceptibility to fluconazole. CONCLUSION: HIV status results in significantly elevated rates of OCC C albicans remains the predominant pathogen, although other species are emerging rapidly. Resistance to fluconazole is on the rise, and more efficient treatment strategies need to be implemented.

Vibrio cholerae O6 gastroenteritis in a patient with lupus nephritis - a report from coastal Karnataka, South India.

INTRODUCTION: Vibrio cholerae O1 strains are responsible for pandemics of cholera and major epidemics in the world. All the remaining V. cholerae non-O1/non-O139 strains are less virulent and are responsible for sporadic cases of gastroenteritis. These non-O1/non-O139 serogroups have more than 200 somatic antigens, and mostly lack cholera toxin and toxin co-regulated pilus encoding genes. Toxigenic and non-toxigenic non-O1/non-O139 V. cholerae have caused several diarrhoeal outbreaks in India and other countries. Acute gastroenteritis is the typical clinical sign and symptom of non-O1/non-O139 V. cholerae infection for both periodical and outbreak cases; in contrast, these V. cholerae are rarely associated with extraintestinal infections. CASE PRESENTATION: Here, we present a case of a 27-year-old female with underlying kidney disease (lupus nephritis) presenting with loose stools, vomiting and fever. V. cholerae O6 was isolated from a faecal sample, which was positive for hlyA and the type III secretion system. The present case is, to the best of our knowledge, the first such case to be reported from South India. CONCLUSION: The V. cholerae O6 associated with autoimmune disease in the present study demonstrates the role of this pathogen in acute gastroenteritis, and if it is left undiagnosed it can lead to septicaemia and other complications. The pathogenic mechanisms of non-O1/non-O139 V. cholerae are multivariate, virulence factors being naturally present in these strains. Therefore, further epidemiological studies are necessary to determine the virulence factors and their pathogenic mechanisms. Non-O1/non-O139 V. cholerae can undoubtedly be the cause of diarrhoea and it would be important to extend bacteriological identification in this line as well as in all cases of gastroenteritis of unknown aetiology.

High primary resistance to metronidazole and levofloxacin, and a moderate resistance to clarithromycin in Helicobacter pylori isolated from Karnataka patients.

BACKGROUND: Due to increased prevalence of H. pylori antimicrobial resistance worldwide and more importantly the resistance patterns vary between different geographical regions, it is important to survey local H. pylori antibiotic resistance profile to provide physicians with more informed drug choices to better treat H. pylori infection. To our knowledge, this is the first study to examine the prevalence of antimicrobial resistance of H. pylori in Karnataka state of South India. RESULTS: A total of 113 H. pylori strains were isolated from gastric biopsies and tested: 81.4% were resistant to metronidazole, 54.9% were resistant to levofloxacin, 20.4% were resistant to clarithromycin, 5.3% were resistant to tetracycline and 7.1% were resistant to amoxicillin. Multidrug resistance was detected in 59.3% of total isolated strains, among which 86.6% were resistant to at least both metronidazole and levofloxacin. In this study, 38 out of 113 H. pylori strains had been whole-genome sequenced. Based on the draft genomes, RdxA and/or FrxA inactivation mutations were found to present in 75% of metronidazole-resistant strains. Clarithromycin-resistant strains had mainly A2143G and G2224A mutations in the 23 rRNA gene. While 87.1% levofloxacin-resistant strains had amino acid substitution mutations occurring predominantly at N87 and D91 in GyrA, novel mutations in the same protein including an insertion of five amino acid residues (QDNSV), immediately after the start codon, and a substitution mutation at R295 were identified. CONCLUSION: High primary resistance to metronidazole and levofloxacin, and a modest occurrence of clarithromycin resistance were revealed in H. pylori strains isolated from Karnataka patients. Therefore metronidazole-, levofloxacin- and clarithromycin-based triple therapies are not suitable as first-line treatment in Karnataka. Both amoxicillin and tetracycline can still be used to eradicate H. pylori infection in this region. We also revealed novel mutations in GyrA protein that possibly contribute to H. pylori resistance in levofloxacin, which merit further investigations.

Epidemiological study of Vibrio cholerae using variable number of tandem repeats.

By conventional genetic methods, including pulse-field gel electrophoresis and multilocus sequence typing, most pathogenic, cholera toxin-positive O1 and O139 isolates of Vibrio cholerae cannot be distinguished. We evaluated relationships among 173 V. cholerae isolates collected between 1992 and 2007 from different geographic areas in India by analyzing five variable number of tandem repeat (VNTR) loci. Each VNTR locus was highly variable, with between 5 and 19 alleles. eburst analysis revealed four large groups of genetically related isolates. Two groups contained genotypes of isolates with the O139 serogroup (which emerged for the first time in epidemic form in 1992), with the other two groups containing O1 strains. In subsequent analysis, it was possible to track the spread of specific genotypes across time and space. Our data highlight the utility of the methodology as an epidemiologic tool for assessing spread of isolates in both epidemic and endemic settings.

Proteinase and phospholipase activity as virulence factors in Candida species isolated from blood.

The number of nosocomial blood stream infections due to Candida species has increased over the past few decades. In order to establish an infection, opportunistic pathogens have to evade the immune system, survive, divide in the host environment, and spread to other tissues. Proteinase and phospholipase secretion has been implicated as potential virulence factors for some Candida species responsible for catheter related candidemia in intensive care unit (ICU) patients with indwelling devices. We therefore have aimed at demonstrating the secretion of proteinase and phospholipase enzymes as virulent factors by Candida species isolated from blood samples collected from ICUs, dialysis units and oncology units. One hundred and fourteen isolates of Candida species were obtained from the blood samples and the isolates include 37 Candida albicans, 7 Candida glabrata, 5 Candida guilliermondii, 3 Candida kefyr, 45 Candida krusei, 5 Candida parapsilosis, and 12 Candida tropicalis. Proteinase assay was performed by using the Staib et al method. Phospholipase assay was performed by using the method of Samaranayake et al. Precipitation zone (Pz value) was determined. The percentage of isolates which produced detectable amounts of proteinase is 74.56% and 44.73% of isolates produced detectable amounts of phospholipase. We believe that production of both phospholipase and proteinase enzimes could be an important virulence factor for several Candida species.

Draft Genome Sequences of 42 Helicobacter pylori Isolates from Rural Regions of South India.

Helicobacter pylori is a successful human gastric pathogen that is associated with the development of gastric cancer. The draft genome sequences of 42 H. pylori clinical strains isolated from South Indian rural populations will provide further insights into the evolution and genetic makeup of Indian H. pylori strains.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serotype Typhimurium Sequence Type 313, Isolated from India.

Salmonella enterica subsp. enterica serotype Typhimurium sequence type 313 (ST313) is most commonly associated with invasive nontyphoidal Salmonella disease in Africa among patients with HIV infection and malignancy. Here, we report a draft genome sequence of S. Typhimurium ST313, isolated from an elderly immunosuppressed patient from India with non-Hodgkins lymphoma.

Paraffin baiting system for demonstration of growth and biofilm production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the commonest pathogens among the pseudomonads. This organism can grow in minimal nutritional requirements. Because of the ability of pseudomonads to grow on paraffin is not commonly found among other human pathogens and the primary human pathogen being P. aeruginosa, we studied the adaptation of this organism to paraffin baiting system for growth and biofilm formation. Strains were tested for the capacity to use paraffin as the sole source of carbon using Czapek's minimal salt medium. Of the 53 clinical isolates of P. aeruginosa, 20 strains exhibited growth by 24 hrs and 42 strains by 48 hrs. The remaining strains did not show any growth in the paraffin baiting system. The oxidase test with the paraffin baiting system was also performed. This simple and inexpensive method can be used to isolate and demonstrate the biochemical and biofilm forming capacity of the organism.