Conventional Microbial Methods Research Articles

Selective formation of high-valent iron in Fenton-like system for emerging contaminants degradation under near-neutral and high-salt conditions

In view of the near-neutral and high-salt conditions, the Fenton technology with hydroxyl radicals (HO•) as the main reactive species is difficult to satisfy the removal of trace emerging contaminants (ECs) in pharmaceutical sewage. Here, a layered double hydroxide FeZn-LDH was prepared, and the selective formation of ≡Fe(IV)=O in Fenton-like system was accomplished by the chemical environment regulation of the iron sites and the pH control of the microregion. The introduced zinc can increase the length of Fe-O bond in the FeZn-LDH shell layer by 0.22 Å compared to that in Fe2O3, which was conducive to the oxygen transfer process between ≡Fe(III) and H2O2, resulting in the ≡Fe(IV)=O formation. Besides, the amphoteric hydroxide Zn(OH)2 can regulate the pH of the FeZn-LDH surface microregion, maintaining reaction pH at around 6.5–7.5, which could avoid the quenching of ≡Fe(IV)=O by H+. On the other hand, owing to the anti-interference of ≡Fe(IV)=O and the near-zero Zeta potential on the FeZn-LDH surface, the trace ECs can also be effectively degraded under high-salt conditions. Consequently, the process of ≡Fe(IV)=O generation in FeZn-LDH system can satisfy the efficient removal of ECs under near-neutral and high-salt conditions.

Chromium (VI) reduction by two-chamber bioelectrochemical system with electrically conductive wall

Hexavalent chromium (Cr(VI)) poses a threat to public health and the environment owing to its high toxicity. Various microbial Cr(VI) reduction methods have been proposed to treat contaminated water; however, they are not effective at high concentrations of Cr(VI), which inhibit microbial growth and viability. To overcome the limitations of conventional microbial methods, we set up a novel bioelectrochemical reactor in which microbial cells were separated from Cr(VI) by an electroconductive barrier consisting of a vertically aligned carbon nanotube (VACNT) composite to remove toxic heavy metal ions. This study demonstrated that chromium compounds could be biologically and sustainably treated with the VACNT composite without growth inhibition, using electron transfer from an electron shuttle secreted by Pseudomonas aeruginosa, even at the microbial inhibitory concentration of Cr(VI). The content of Cr(VI) was measured using the diphenylcarbazide method, and Cr(III), the reduction product, was identified using X-ray photoelectron spectroscopy. Pyocyanin, a phenazine secreted by P. aeruginosa, was identified as an electron mediator in Cr(VI) reduction, using aeration and phz mutant assay. The bioelectrochemical reactor used in this study exhibited a Cr(VI) removal rate of 99.6% at 100 mg/L, which is the highest reduction rate achieved using P. aeruginosa. The microbial Cr(VI) reduction system developed in this study allows microbial Cr(VI) reduction to overcome the hurdles of biological treatment of toxic materials and has advanced the understanding of microbial electron transfer.

Novel members of bacterial community during a short-term chilled storage of common carp (Cyprinus carpio)

This work aimed to identify the key members of the bacterial community growing on common carp (Cyprinus carpio) fillets during chilled storage with next-generation sequencing (NGS) and cultivation-dependent methods. Carp fillets were stored for 96 h at 2 °C and 6 °C with and without a vacuum package, and an additional frozen-thawed storage experiment was set for 120 days. Community profiles of the initial and stored fish samples were determined by amplicon sequencing. Conventional microbial methods were used parallelly for the enumeration and cultivation of the dominant members of the microbial community. Cultivated bacteria were identified with 16S rRNA sequencing and the MALDI-TOF MS method. Based on our results, the vacuum package greatly affected the diversity and composition of the forming microbial community, while temperature influenced the cell counts and consequently the microbiological criteria for shelf-life of the examined raw fish product. Next-generation sequencing revealed novel members of the chilled flesh microbiota such as Vagococcus vulneris or Rouxiella chamberiensis in the vacuum-packed samples. With traditional cultivation, 161 bacterial strains were isolated and identified at the species level, but the identified bacteria overlapped with only 45% of the dominant operational taxonomic units (OTUs) revealed by NGS. Next-generation sequencing is a promising and highly reliable tool recommended to reach a higher resolution of the forming microbial community of stored fish products. Knowledge of the initial microbial community of the flesh enables further optimization and development of processing and storage technology.

MICROBIOLOGICAL QUALITY OF MOROCCAN LABELED EUPHORBIA RESINIFERA HONEY

In the present work, microbiological profile of thirty-seven samples of labeled honey were collected in a Protected Geographical Indication “PGI” area of Tadla-Azilal region, which is an endemic zone of Euphorbia resinifera plant. A profile was assessed using conventional microbial methods, like enumeration, detection and/or germs identification, in accordance with ISO norms. This is the first study in which a honey with Moroccan “PGI” was tested, in order to assess its compliance with bacteriological recommendations. Coliforms (Total and fecal Coliforms), Salmonella spp., Shigella spp., Sporus of Bacillus cereus and Clostridium perfringens were not detected. The numbers of Standard Plate Count “SPC” were less than 102 CFU.g-1 for all samples. The molds and yeasts were found among samples and 32% and 40% of samples were positive, respectively. However, no samples showed a higher value than recommended limit [102 CFU.g-1]. We conclude that samples of labeled euphorbia honey of Tadla-Azilal analyzed present good commercial quality parameters (SPC, molds and yeasts “absence of unwanted fermentations”), a good sanitary quality (absence of coliforms and S. aureus) and are safe (Slam., Shig., Sporus of B. cereus and C. perf.). Standardization (regulation and specifications) and a rationalization of beekeeping techniques throughout Euphorbia “PGI” area studied may further sustainably improve the quality of this unique honey, and ensure it over the years.

Antimicrobial profile of Salmonella enterica serotype Choleraesuis from free-range swine in Kakamega fish market, western Kenya.

Salmonella enterica subspecies enterica serovar Choleraesuis is a host-adapted, facultative, intracellular pathogen that causes swine paratyphoid. Its antimicrobial resistance presents a challenge to feed manufacturing industries. However, stopping antibiotics in animal feed would have economic implications for the industry. Conventional microbial methods for isolation and identification of S. Choleraesuis were employed. The isolates were subjected to screening against 17 antimicrobial agents and genotyping of resistance markers by PCR. The data were then analyzed and presented in percentages. Phenotypically, 43 out of 95 isolates showed multidrug resistance. Among the 17 antibiotics tested, resistance was observed as follows: sulphonamides (45.2%), nalidixic acid (44.25%), tetracycline (42%), ampicillin (36.8%), erythromycin (34.7%), carbenicillin (31.5%), chrolamphenical (28.4%), gentamicin (27.3%), kanamycin (24.2%), spectinomycin (21%), sulfamethoxazole-trimethoprim (16.8%), streptomycin (12.6%), cephalothion (8.4%), ofloxacin (5.2%), ciprofloxacin (4.2%), and norfloxacin (4.2%). Fifty-two isolates were susceptible to the antimicrobial agents tested. A total of 3.1% of the isolates had the integron gene pattern combination of dfrA2-aadA2 (2100 bp), dfrA12 (2100 bp); 4.2% had dfrA12-aadA2-sulI (2100 bp); 2.1% had dfrA12-aadA2 (2100 bp); and 1% had dfrA2-aadA2-sulI (2100 bp), oxa1-aadA2 (1500 bp), dfrA12-aadA2-sulI, and blaPSE (2100 bp). The isolated S. Choleraesuis were resistant to more than 10% of the antimicrobial agents used in this study. Appropriate surveillance is warranted to gain more information about the epidemiology, as stopping antibiotics in animal feed would have economic implications for the industry.

A microbiology-based multi-parametric approach towards assessing biological stability in drinking water distribution networks

Biological stability of drinking water implies that the concentration of bacterial cells and composition of the microbial community should not change during distribution. In this study, we used a multi-parametric approach that encompasses different aspects of microbial water quality including microbial growth potential, microbial abundance, and microbial community composition, to monitor biological stability in drinking water of the non-chlorinated distribution system of Zürich. Drinking water was collected directly after treatment from the reservoir and in the network at several locations with varied average hydraulic retention times (6–52 h) over a period of four months, with a single repetition two years later. Total cell concentrations (TCC) measured with flow cytometry remained remarkably stable at 9.5 (± 0.6) × 104 cells/ml from water in the reservoir throughout most of the distribution network, and during the whole time period. Conventional microbial methods like heterotrophic plate counts, the concentration of adenosine tri-phosphate, total organic carbon and assimilable organic carbon remained also constant. Samples taken two years apart showed more than 80% similarity for the microbial communities analysed with denaturing gradient gel electrophoresis and 454 pyrosequencing. Only the two sampling locations with the longest water retention times were the exceptions and, so far for unknown reasons, recorded a slight but significantly higher TCC (1.3 (± 0.1) × 105 cells/ml) compared to the other locations. This small change in microbial abundance detected by flow cytometry was also clearly observed in a shift in the microbial community profiles to a higher abundance of members from the Comamonadaceae (60% vs. 2% at other locations). Conventional microbial detection methods were not able to detect changes as observed with flow cytometric cell counts and microbial community analysis. Our findings demonstrate that the multi-parametric approach used provides a powerful and sensitive tool to assess and evaluate biological stability and microbial processes in drinking water distribution systems.

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.

Bio-safety Assessment of Validamycin Formulation on Bacterial and Fungal Biomass in Soil Monitored by Real-Time PCR

Validamycin is a biological fungicide used in the agriculture sector. It can control sheath blight caused by Rhizoctonia solani in rice, potatoes, vegetables, as well as damping off diseases in vegetable seedlings, cotton, sugar beets and other plants on a large scale (NIOSH, 1993). It is absorbed by cells of Rhizoctonia solani and hydrolyzed to a potent inhibitor of trehalase-validoxylamine A. Validamycin has been considered as a low-toxicity material that can be degraded easily by some bacterium, such as Flavobacterium saccharophilum, Pseudomonas sp. HZ519 and Stenotrophomonas maltrophilia CCTCC M 204024 under lab conditions (Asano et al., 1984; Zheng et al., 2005; zhang et al., 2005). To the best of our knowledge, few studies have focused on the effects of validamycin formulation on the agro-ecosystem. Crystal proteins of Bacillus thuringiensis (Bt) also serve as a biological pesticide, also regarded as non-toxic several decades ago. However, following the emergence of Bt-transgenic plants, some reports showed that Bt toxins could accumulate in the soil, resulting in potential risks to soil biological processes including microorganism biomass and soil enzyme activity (Castaldini et al., 2005; Donegan et al., 1995; Wang et al., 2005; Wu et al., 2004a, b). In essence, a biological pesticide is a chemical substance with a definite molecular weight, and can also cause environmental disturbance, just like chemical pesticides (Shen, 1997). Bearing in mind these concerns, it is necessary to assess the effects of validamycin formulation on agro-ecosystem and the corresponding risks. The widespread and nonjudicious agricultural use of pesticides results in their entry into the soil and water ecosystems. Soil is the most diverse terrestrial habitat, and soil microbial communities play important roles in decomposition, nutrient cycling, and energy flow (Waldrop and Firestone, 2006; Wardle and Giller, 1997). Much work has been directed towards understanding the complexity of pesticide–microbial interactions in soil, indicating that in general soil microbial populations are affected by pesticide (Griffiths et al., 2006; Lu et al., 2006). Conventional microbial methods that rely on microbial cultivation can only identify a small fraction (0.01–10%) of the total microbial biomass (Ferguson et al., 1984), because they are unable to culture the majority of microorganisms from soil samples. This was a main obstacle for the better understanding of microbial ecology and diversity. Recently, soil microbiologists have developed several molecular methods to overcome this limitation by allowing particular genes to be monitored directly in the environment. Such methods include the polymerase chain reaction (PCR), and microarray-based genomic technology. Real-time PCR is a method based on the use of fluorescent probes or dyes to quantify the copy number of target DNA in a sample. This technology, which is not limited by the cultivability of soil microorganisms, has been successfully applied in the medical and environmental fields for the quantification of bacteria (Kolb et al., 2003), viruses (Schaade et al., 2000) and fungi (Lees et al., 2002). In this investigation, we developed a real-time PCR assay that can quantify soil bacteria and fungi by using specific primers based on 16S H. Qian (&) D. Cao W. Chen X. Xu Y. Lu College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, China e-mail: hfqian@126.com

Is Streptococcus Pneumoniae the leading cause of pneumonia of unknown etiology? a microbiologic study of lung aspirates in consecutive patients with community-acquired pneumonia

PURPOSE: Although a wide variety of recognized pathogens can cause community-acquired pneumonia, in many patients the etiology remains unknown after routine diagnostic workup. The aim of this study was to identify the causal agent in these patients by obtaining lung aspirates with transthoracic needle aspiration. SUBJECTS AND METHODS: During a 15-month period, all consecutive patients with community-acquired pneumonia who were eligible for transthoracic needle aspiration were enrolled in the study. In addition to conventional microbial methods (culture of blood and sputum, serologic studies), we performed cultures and genetic and antigen tests for common respiratory pathogens in lung aspirates. RESULTS: The study group consisted of 109 patients. Conventional microbial studies identified an etiology in 54 patients (50%), including Mycoplasma pneumoniae in 19 patients, Chlamydia pneumoniae in 9 patients, and Streptococcus pneumoniae in 9 patients. Among the remaining 55 patients, study of the lung aspiration provided evidence of the causal agent in 36 (65%). In 4 additional patients with a single microbial diagnosis by conventional methods, the lung sample provided evidence of an additional microorganism. The new pathogens detected by lung aspiration were S. pneumoniae in 18 patients, Haemophilus influenzae in 6 patients, Pneumocystis carinii in 4 patients, and C. pneumoniae in 3 patients; other organisms were identified in 4 patients. CONCLUSIONS: In our study, S. pneumoniae was the leading cause of community-acquired pneumonia, accounting for 25% of all cases, including about one-third of the cases the cause of which could not be ascertained with routine diagnostic methods.

Investigation of bacterial infections of the lower respiratory tract

Conventional microbial methods can yield an aetiological diagnosis in almost all of the patients admitted with community-acquired pneumonia. Sputum examination, if accurately performed, is still one of the most important diagnostic tools. Antigen detection, especially for pneumococcal antigen, is a valuable and rapid method for diagnosing pneumococcal pneumonia even during antibiotic treatment. Because of the high fatality rate in severe community-acquired pneumonia, invasive procedures may be indicated and are useful; they are preferably performed before antibiotic treatment is initiated. Bronchoalveolar lavage has the advantage of sampling secretions from a relatively large part of the lobe, and this fulfils the requirements for appropriate specimens for all microbiological procedures. Polymerase chain reaction is the most promising new diagnostic technique in pulmonary infections. It can detect DNA or RNA of a low number of microorganisms with high accuracy in different biological fluids. Larger studies are necessary to investigate the impact of this technique on the aetiology, treatment and outcome of community-acquired pneumonia. Although serology is useful for aetiological and epidemiological studies, in the majority of cases the results of serology are available only after completion of treatment, and therefore seldom influence the choice of specific antibiotic.