Bacterial Panel Research Articles

Isolation and Characterization of Multidrug-Resistant Gram-Negative Bacteria Found in Free-Ranging Long-Eared Hedgehogs (Erinaceus concolor) From Tabriz, Iran

Hedgehogs, in particular the African Hedgehog (Atelerix albiventris), are gaining acceptance as a companion animal; however, information regarding their normal bacterial flora is limited. Hedgehogs are common and widely distributed in urban and rural areas in Iran. The objectives of this study were to identify the Gram-negative bacteria in the gastrointestinal tract (GIT) of free-ranging long-eared hedgehogs (Erinaceus concolor) and determine specific antibiotic susceptibility of the isolates. Culture samples were collected from different areas of the GIT, including the small and large intestine, and then submitted for culture. The bacterial sensitivity panel consisted of ampicillin (10MU/g), amoxicillin-clavulanate (30MU/g), cefixime (5MU/g), polymyxin E (10MU/g), ceftriaxone (30MU/g), ciprofloxacin (5MU/g), chloramphenicol (30MU/g), gentamicin (10MU/g), kanamycin (30MU/g), and tetracycline (30MU/g). A total of 58 Gram-negative bacteria, consisting of 8 genera, were isolated from the long-eared hedgehogs including Escherichia coli (34.5%), Proteus (27.5%), Pseudomonas (12%), Shigella (8.7%), Yersinia (6.9%), Salmonella (6.9%), and Klebsiella (3.4%). Antimicrobial susceptibility testing using the Kirby-Bauer disk diffusion method showed that 24 isolates (41.3%) exhibited resistance phenotypes to multiple antimicrobial agents. Our results indicate that free-ranging long-eared hedgehogs have primarily a Gram-negative population of bacteria in the GIT.

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.

Antimicrobial activity and toxicity of Syzygium australe and Syzygium leuhmannii fruit extracts

Introduction: Many species of Syzygium are known to have antiseptic activity. Several Australian Syzygium species had roles as traditional bush medicines for Australian Aborigines although their antiseptic potential has not been rigorously studied. Methods: The antimicrobial activity of solvent extracts of Syzygium australe and Syzygium leuhmannii fruits were investigated by disc diffusion assay against a panel of bacteria and fungi and their MIC values were determined. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: The methanolic extracts of the fruit of both Syzygium species displayed the greater antibacterial activity of the extracts tested. S. australe generally had greater efficacy than the S. leuhmannii extracts. S. australe and S. leuhmannii fruit methanolic extract inhibited the growth of 13 (93%) and 12 (86%) of the 14 bacteria tested. Gram-positive and Gram-negative bacteria were both susceptible, although a slightly greater susceptibility of Gram-positive bacteria was noted. Nine (90%) and 8 (80%) of the 10 Gram-negative bacteria had their growth inhibited by S. australe and S. leuhmannii fruit methanolic extract extracts respectively. In contrast, methanolic extracts of both species inhibited growth of 100% of the Gram-positive bacteria tested. None of the extracts displayed broad antifungal activity. Indeed, none of the extracts inhibited the growth of A. niger or C. albicans. Only S. cerevisiae growth was affected and then only by the chloroform and hexane extracts. The methanolic, aqueous and ethyl acetate extracts of both Syzygium species were toxic in the Artemia franciscana bioassay, inducing significant mortality at <1000 µg/ml. Conclusions: The inhibitory bioactivity of S. australe against the bacterial panel validate Australian Aboriginal usage of S. australe leaves as antiseptic agents and confirms their medicinal potential, although care is needed in the uses of these extracts for these purposes due to their reported toxicity.

Comparison of the BD MAX Enteric Bacterial Panel to Routine Culture Methods for Detection of Campylobacter, Enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella Isolates in Preserved Stool Specimens

We compared the BD MAX enteric bacterial panel (EBP) to culture for the detection of Salmonella, Shigella, Campylobacter, and Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157 in seeded stool samples. The EBP panel demonstrated superior sensitivity and reliably detected Salmonella, EHEC O157, Shigella, and Campylobacter at concentrations 1- to 2-log10 lower than those needed for culture detection.

Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels

We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP(-)/rtPCR(+) colonies were identified as C. perfringens, whereas 3 mCP(+)/rtPCR(-) colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

Broad Spectrum Antimicrobial Activity of Melimine Covalently Bound to Contact Lenses

To develop a stable antimicrobial contact lens, which is effective against the International Organization for Standardization (ISO) panel microorganisms, Acanthamoeba castellanii and drug resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus. Melimine was covalently incorporated into etafilcon A lenses. The amount of peptide present on the lens surface was quantified using amino acid analysis. After coating, the heat stability (121°C), lens surface hydrophobicity (by captive bubble), and in vitro cytotoxicity to mouse L929 cells of the lenses were investigated. Antimicrobial activity against the micro-organisms was evaluated by viable plate count and fluorescence microscopy, measuring the proportion of cell death compared with control lenses with no melimine. The most effective concentration was determined to be 152 ± 44 μg lens(-1) melimine on the lens surface. After coating, lenses were relatively hydrophilic and were nontoxic to mammalian cells. The activity remained high after autoclaving (e.g., 3.1, 3.9, 1.2, and 1.0 log inhibition against P. aeruginosa, S. aureus, A. castellanii, and Fusarium solani, respectively). Fluorescence microscopy confirmed significantly reduced (P < 0.001) adhesion of viable bacteria to melimine contact lenses. Viable count confirmed that lenses were active against all the bacteria and fungi from the ISO panel, Acanthamoeba and gave at least 2 log inhibition against all the multidrug resistant S. aureus and P. aeruginosa strains. Melimine may offer excellent potential for development as a broad spectrum antimicrobial coating for contact lenses, showing activity against all the bacterial and fungal ISO panel microorganisms, Acanthamoeba, and antibiotic resistant strains of P. aeruginosa and S. aureus.

A bacterial bioreporter panel to assay the cytotoxicity of atmospheric particulate matter

Numerous studies have demonstrated that elevated concentrations of suspended atmospheric particulate matter (PM) are associated with adverse health effects. In order to minimize the adverse public health effects of atmospheric PM by exposure management, there is a need for a greater understanding of the toxic mechanisms and the components that are responsible for the toxic effects. The aim of this study was to utilize bioassay techniques to investigate these aspects. For this purpose a reporter panel of 9 genetically engineered bacterial (Escherichia coli) strains was composed. Each panel member was designed to report on a different stress condition with a measurable light signal produced by the luciferase enzyme.Toxic mechanisms and components were studied using six anthropogenic PM source samples, including two vehicle combustion particles, three coal fly ash (CFA) samples and an urban dust sample. The most prominent outcome of the panel exposure results were broad panel responses observed for two of the CFA samples, indicating oxidative stress, respiration inhibition and iron deficiency. These responses were relieved when the samples were treated with EDTA, a non-specific metal chelator, suggesting the involvement of metals in the observed effects. Bioavailability analysis of the samples suggests that chromium was related to the toxic responses induced by two of the CFA samples. Oxidative stress was also observed in several samples of ambient atmospheric aerosols and excess metal toxicity in an urban dust sample collected in a parking lot.The reporter panel approach, as demonstrated in this study, has the potential of providing novel insights as to the mechanisms of atmospheric PM toxicity. Furthermore, combining the panel's results with bioavailability data can enlighten about the role of different PM components in the observed toxicity.

Antimicrobial Activity of Syzygium australe and Syzygium leuhmannii Leaf Methanolic Extracts

ABSTRACT Introduction: Many species of Syzygium are known to have antiseptic activity. Australian Syzygium species had roles as traditional bush medicines for Australian Aborigines although their antiseptic potential has not been rigorously studied. Methods: The antimicrobial activity of methanolic extracts of Syzygium australe and Syzygium leuhmannii leaves was investigated by disc diffusion assay and growth time course assay against a panel of bacteria and fungi. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: S. australe leaf extract inhibited the growth of 10 of the 14 bacteria tested (71%). Gram-positive and Gram-negative bacteria were equally susceptible. 8 of the 11 Gram-negative bacteria (73%) and 2 of the 3 Gram-positive bacteria (67%) tested had their growth inhibited by S. australe leaf extract. The extract also displayed antifungal activity against a nystatin resistant strain of A. niger but did not affect C. albicans or S. cerevisiae growth. In comparison, S. leuhmannii leaf extract did not inhibit the growth of any of the microbial agents tested in the disc diffusion assay. The antibacterial activity of S. australe leaf extract was further investigated by growth time course assays which showed significant growth inhibition in cultures of A. hydrophilia, B. cereus, and P. fluorescens within 1 h but not of B. subtilis. S. leuhmannii also inhibited the growth of P. fluorescens and to a lesser extent, A. hydrophilia in the time course assay. Both Syzygium extracts displayed low toxicity in the Artemia franciscana bioassay. Conclusions: The low toxicity of these Syzygium extracts and the inhibitory bioactivity of S. australe against the bacterial panel validate Australian Aboriginal usage of S. australe leaves as antiseptic agents and confirms their medicinal potential.

A bacterial reporter panel for the detection and classification of antibiotic substances.

SummaryThe ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.

Development of Hydrolysis Probe-Based Real-Time PCR for Identification of Virulent Gene Targets ofBurkholderia pseudomalleiandB. mallei—A Retrospective Study on Archival Cases of Service Members with Melioidosis and Glanders

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

Multi-Probe Real-Time PCR Identification of Common Mycobacterium Species in Blood Culture Broth

Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use.

A real-time PCR assay for the detection of Neisseria gonorrhoeae in genital and extragenital specimens

A Neisseria gonorrhoeae LightCycler (NGpapLC) assay targeting the porA pseudogene was compared with bacterial culture for detection of N. gonorrhoeae in 636 clinical specimens (216 cervical, 185 urethral, 196 throat, and 39 rectal swab specimens). The specificity of the NGpapLC assay was further investigated by testing a bacterial reference panel comprising several Neisseria species. Overall, 19 (3.0%) specimens were positive and 613 (96.4%) specimens were negative by both methods. Four (0.6%) specimens were positive by the NGpapLC assay only. For the cervical and urethral swabs, the NGpapLC provided 100% sensitivity and 100% specificity compared with bacterial culture. Following discrepant analysis, the clinical sensitivity and specificity of the NGpapLC for throat and rectal swabs was also 100%. For the bacterial panel, only N. gonorrhoeae isolates provided positive results. The results show the NGpapLC assay is suitable for use on a range of clinical specimens and could improve detection of pharyngeal N. gonorrhoeae.

Development of Highly Specific Monoclonal Antibodies for the Diagnosis ofVibrio cholerae01

We report here the development of two monoclonal antibodies, termed 5G8 and 5C12, belonging to the IgM and IgG1 class, respectively, suitable for the identification of Vibrio cholerae 01 in clinical and environmental samples. The specificities of the monoclonals were evaluated by ELISA and indirect immunofluorescent microscopy of microorganisms normally present in stool samples and with two bacterial panels. One panel included 72 potentially antigenically related bacterial strains and the second panel included 20 pathogenic bacterial strains involved in diarrhea cases. The results of these extensive analyses indicate that monoclonal antibodies 5G8 and 5C12 are highly specific and suitable for the clinical diagnosis of Vibrio cholerae 01 in human stool samples by indirect immunofluorescent microscopy. Although the antigenic sites recognized by these antibodies were not identified in this study, the observation of Western blot patterns suggested that 5G8 and 5C12 monoclonal antibodies bind to LPS epitopes, a good structural marker for the detection of V. cholerae 01 because it is present in all bacterial cell walls.