Environmental Bacterial Strains Research Articles

Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans.

In the context of the great concern about the impact of human activities on the environment, we studied 403 commensal Escherichia coli/Escherichia clade strains isolated from several animal and human populations that have variable contacts to one another. Multilocus sequence typing (MLST) showed a decrease of diversity 1) in strains isolated from animals that had an increasing contact with humans and 2) in all strains that had increased antimicrobial resistance. A specific B1 phylogroup clonal complex (CC87, Institut Pasteur schema nomenclature) of animal origin was identified and characterized as being responsible for the increased antimicrobial resistance prevalence observed in strains from the environments with a high human-mediated antimicrobial pressure. CC87 strains have a high capacity of acquiring and disseminating resistance genes with specific metabolic and genetic determinants as demonstrated by high-throughput sequencing and phenotyping. They are good mouse gut colonizers but are not virulent. Our data confirm the predominant role of human activities in the emergence of antimicrobial resistance in the environmental bacterial strains and unveil a particular E. coli clonal complex of animal origin capable of spreading antimicrobial resistance to other members of microbial communities.

Sources of antibiotics in natural environments and their biological role

Nowadays antibiotics are broadly used not only for treatment of bacterial infections but also in nonmedical applications. For many years they have been added as livestock and poultry growth supplements, and they are applied similarly in fish farming. In basically unchanged form they may get into the natural environment and remain there for a long time. Excessive use of antibiotics leads to widespread of antibiotic resistance among clinical and environmental bacterial strains. Subinhibitory concentrations of antibiotics, which do not inhibit growth of bacteria, are often found in soil, water or even in the tissue of different organisms. Such low concentrations affect many bacterial genes through changes in their transcription level and increase of the mutation rate, and as a consequence lead to many bacterial adaptations to environmental stresses. There is also evidence that subinhibitory concentrations of antibiotics induce transfer of mobile genetic elements through horizontal gene transfer pathways, and therefore enhance antibiotic resistance, also among environmental strains. The analyzed data suggest the necessity of restriction and regular monitoring of antibiotics, which may be considered as environmental pollutants.

Genetic structure and biological properties of the first ancient multiresistance plasmid pKLH80 isolated from a permafrost bacterium.

A novel multidrug-resistance plasmid, pKLH80, previously isolated from Psychrobacter maritimus MR29-12 found in ancient permafrost, was completely sequenced and analysed. In our previous studies, we focused on the pKLH80 plasmid region containing streptomycin and tetracycline resistance genes, and their mobilization with an upstream-located ISPpy1 insertion sequence (IS) element. Here, we present the complete sequence of pKLH80 and analysis of its backbone genetic structure, including previously unknown features of the plasmid's accessory region, notably a novel variant of the β-lactamase gene blaRTG-6. Plasmid pKLH80 was found to be a circular 14 835 bp molecule that has an overall G+C content of 40.3 mol% and encodes 20 putative ORFs. There are two distinctive functional modules within the plasmid backbone sequence: (i) the replication module consisting of repB and the oriV region; and (ii) the mobilization module consisting of mobA, mobC and oriT. All of the aforementioned genes share sequence identities with corresponding genes of different species of Psychrobacter. The plasmid accessory region contains antibiotic resistance genes and IS elements (ISPsma1 of the IS982 family, and ISPpy1 and ISAba14 of the IS3 family) found in environmental and clinical bacterial strains of different taxa. We revealed that the sequences flanking blaRTG-6 and closely related genes from clinical bacteria are nearly identical. This fact suggests that blaRTG-6 from the environmental strain of Psychrobacter is a progenitor of blaRTG genes of clinical bacteria. We also showed that pKLH80 can replicate in different strains of Acinetobacter and Psychrobacter genera. The roles of IS elements in the horizontal transfer of antibiotic resistance genes are examined and discussed.

An epifluorescence-based evaluation of the effects of short-term particle association on the chlorination of surface water bacteria

Investigations into particle-mediated chlorination resistance were undertaken for three different bacteria (Escherichia coli ATCC 25922 and environmental isolates of Pseudomonas fluorescens and Serratia marcescens) and three different surfaces (goethite, environmental particles and surface-modified environmental particles). P. fluorescens demonstrated greater hydrophobicity than both other strains and proved the most adherent bacterium over all substrata investigated. Particle-mediated resistance to chlorination was investigated using short bacteria-particle association times and activity assays that employed sensitive epifluorescent detection. Consistent with adhesive behaviours, the bacterial strain that demonstrated the greatest particle-mediated chlorination resistance was the environmental strain of P. fluorescens. Resistance was observed to vary with both bacteria and particle type, and demonstrated a moderate correlation with adhesion (r2 ≥ 0.65). The short-term approach employed in our study demonstrates particle-mediated protection without the commonly assumed requirements of extracellular polymeric substances (EPS) or a large particle-based chlorine demand. Consequently, we have linked resistance with adhesion capacities and demonstrated a limit to resistance in the presence of additional particle protective sites (through increased turbidity) which appears to be driven by intra-population variance in bacterial surface characteristics. Finally, we observed important differences between behaviours of environmental versus laboratory-derived bacterial strains and particles, which highlight the importance of employing both approaches in characterising “real world” systems.

Contrasting responses of marine bacterial strains exposed to carboxylated single-walled carbon nanotubes

The potential toxic effects of carboxylated (COOH) single-walled carbon nanotubes (SWNTs) were investigated on the cell growth and viability of two reference (Silicibacter pomeroyi, Oceanospirillum beijerinckii) and two environmental (Vibrio splendidus, Vibrio gigantis) Gram-negative marine bacterial strains. Bacterial cells were exposed to six concentrations of SWNT-COOH, during different incubation times. Our results revealed different sensitivity levels of marine bacterial strains toward SWNT-COOH exposure. A bactericidal effect of SWNT-COOH has been observed only for Vibrio species, with cell loss viability estimated to 86% for V. gigantis and 98% for V. splendidus exposed to 100μgmL−1 of SWNT-COOH during 2h. For both Vibrio strains, dead cells were well individualized and no aggregate formation was observed after SWNT-COOH treatment. The toxic effect of SWNT-COOH on O. beijerinckii cells displayed time dependence, with a longer exposure time reducing their specific growth rate by a factor of 1.2. No significant effect of SWNT-COOH concentration or incubation time had been demonstrated on both growth ability and viability of S. pomeroyi, suggesting a stronger resistance capacity of this strain to carbon nanotubes. The analysis of the relative expression of some functional genes involved in stress responses, using the real-time reverse transcriptase PCR, suggests that the cell membrane damage is not the main toxicity mechanism by which SWNT-COOH interacts with marine bacterial strains. Overall, our results show that SWNT-COOH present a strain dependent toxic effect to marine bacteria and that membrane damage is not the main toxicity mechanism of SWNT in these bacteria.

Isolation and Bioactivity Screening of Marine Environmental Bacteria from Trachurus Murphyi Chile Fishery

Marine bacteria have been proven to be a profound resource on the development of natural product chemistry and upon the medical sciences. The pelagic fishery locates the wide international public sea regions and contains huge resource and technical benefits. The increasingly greater potential for exploitation of new marine microbes resource in these special sea areas has become conspicuous. In this paper, the marine environmental bacterial strains were isolated from water sampled from the Trachurus murphyi Chile fishery in southeast Pacific Ocean, and the anti-bacterial bioactivity screening for the crude extract from the strain fermentation, and taxonomic identification of the screened bioactive strains were then performed. The obtained result in this study has showed that marine environmental bacterial isolated from Trachurus murphyi Chile fishery demonstrated good potential for the further exploration for marine bioactive natural products.

Antibacterial activity of some selected 1,2,4-triazole derivatives against standard, environmental, and medical bacterial strains

Recently, four heterocyclic compounds containing 1,2,4-triazole (designated as Y2, Y3, Y30, and Y32) were synthesized by our lab. Previous data showed that 1,2,4-triazole nucleus posses a wide range of pharmacological activities such as antioxidant, antiviral, antitumor and antibacterial activities. Currently, the need for triazole derivatives with a broad spectrum of antibacterial activity are in the rise. Therefore, these data encouraged us to test newly synthesized 1,2,4-triazole compounds for their antibacterial activity against three types of bacteria: standard, environmental and medical bacteria. Both well diffusion and broth dilution methods were employed to examine the potential antibacterial activity against the aforementioned types of bacteria. The standard bacteria include Staphylococcus aureus ATCC 29213, Staphylococcus epidermis ATCC 12228, and Bacillus

Photodegradation of fluoroquinolones in surface water and antimicrobial activity of the photoproducts

The widespread presence of fluoroquinolone antibiotics (FQs) in natural ecosystems is a health hazard for humans and other living organisms. The role of sunlight in degrading FQs present in environmental waters has been studied. In particular, the photodegradation of four largely employed FQs, viz. Ciprofloxacin (CIP), Danofloxacin (DAN), Levofloxacin (LEV) and Moxifloxacin (MOX) has been studied in not tampered river water. Degradation rates have been investigated at ppb levels (20–50 μg L−1) under solar light, and the results have been commented critically. The products distribution has been studied by HPLC-ESI-MS/MS analysis and structures have been attributed on the basis of their mass fragmentation spectra.Importantly from the environmental point of view, the (potentially toxic) FQ nucleus remained intact over the early stages of the degradation. Indeed, the photoproducts were proved to possess residual antibacterial activity, as shown from in vitro antibacterial activity tests against different well characterized human and environmental bacterial strains, carried out on the above FQs, as well as for Enrofloxacin (ENR) and Marbofloxacin (MAR).

Electroactivity of Phototrophic River Biofilms and Constitutive Cultivable Bacteria

Electroactivity is a property of microorganisms assembled in biofilms that has been highlighted in a variety of environments. This characteristic was assessed for phototrophic river biofilms at the community scale and at the bacterial population scale. At the community scale, electroactivity was evaluated on stainless steel and copper alloy coupons used both as biofilm colonization supports and as working electrodes. At the population scale, the ability of environmental bacterial strains to catalyze oxygen reduction was assessed by cyclic voltammetry. Our data demonstrate that phototrophic river biofilm development on the electrodes, measured by dry mass and chlorophyll a content, resulted in significant increases of the recorded potentials, with potentials of up to +120 mV/saturated calomel electrode (SCE) on stainless steel electrodes and +60 mV/SCE on copper electrodes. Thirty-two bacterial strains isolated from natural phototrophic river biofilms were tested by cyclic voltammetry. Twenty-five were able to catalyze oxygen reduction, with shifts of potential ranging from 0.06 to 0.23 V, cathodic peak potentials ranging from -0.36 to -0.76 V/SCE, and peak amplitudes ranging from -9.5 to -19.4 μA. These isolates were diversified phylogenetically (Actinobacteria, Firmicutes, Bacteroidetes, and Alpha-, Beta-, and Gammaproteobacteria) and exhibited various phenotypic properties (Gram stain, oxidase, and catalase characteristics). These data suggest that phototrophic river biofilm communities and/or most of their constitutive bacterial populations present the ability to promote electronic exchange with a metallic electrode, supporting the following possibilities: (i) development of electrochemistry-based sensors allowing in situ phototrophic river biofilm detection and (ii) production of microbial fuel cell inocula under oligotrophic conditions.

Antibiotic Producing Potentials of Three Freshwater Actinomycetes Isolated from the Eastern Cape Province of South Africa

Crude extracts of three actinomycetes species belonging to Saccharopolyspora (TR 046 and TR 039) and Actinosynnema (TR 024) genera were screened for antibacterial activities against a panel of several bacterial strains. The extracts showed antibacterial activities against both gram-negative and gram-positive test bacteria with inhibition zones ranging from 8 to 28 mm (TR 046); 8 to15 mm (TR 039); and 10 to 13 mm (TR 024). The minimum inhibitory concentrations ranged from 0.078 to 10 mg/mL (TR 046); 5 to >10 mg/mL (TR 039); and 1.25 to 5 mg/mL (TR 024). Time-kill studies revealed that crude extract of TR 046 showed strong bactericidal activity against Bacillus pumilus (ATCC14884), reducing the bacterial load by 104 cfu/mL and 102 cfu/mL at 4× MIC and 2× MIC, respectively, after 6 h of exposure. Similarly, against Proteus vulgaris (CSIR 0030), crude extract of TR 046 achieved a 0.9log10 and 0.13log10 cfu/mL reduction at 5 mg/mL (4× MIC) and 1.25 mg/mL (2× MIC) after 12 h of exposure. The extract was however weakly bactericidal against two environmental bacterial strains (Klebsiella pneumoniae and Staphylococcus epidermidis); and against Pseudomonas aeruginosa (ATCC 19582): the extract showed bacteriostatic activities at all concentrations tested. These freshwater actinomycetes appear to have immense potential as a source of new antibacterial compound(s).

Toxicity of CdTe Quantum Dots in Bacterial Strains

Contradictory results on quantum dot cytotoxicity exist for many types of biological systems, especially microorganisms. In this study, we compare the cytotoxicity of CdTe quantum dots (QDs) to four very different environmental bacterial strains, giving quantitative models of the growth curves for exposed organisms. The mechanisms of toxicity are explored by measuring reactive oxygen species generation by the QDs alone and investigating the oxidative damage to mutant bacteria especially sensitive to ROS. Electron microscopic examination also reveals factors that may contribute to resistance to nanoparticles in some strains.

Effect of pH on cadmium and lead binding by extracellular polymeric substances (EPS) extracted from environmental bacterial strains

Cd and Pb binding by extracellular polymeric substances (EPS) extracted from seven environmental bacterial strains were investigated as a function of pH. The study was carried out by using a polarographic method in the stripping mercury dropping electrode (SMDE) mode which is able to determine labile metal in solution containing soluble ligands such as EPS. The results obtained provide evidence for the presence of a pH-sorption/desorption edge for Cd and Pb by EPS. Kurbatov's model gives information on the mechanisms involved through the determination of “relative complexation constants” (operationally defined) and the number of exchanged protons. This model shows that proton exchange with metals is not the only mechanism involved in metal biosorption by bacterial EPS. The position of the pH-sorption edge curves and the “relative complexation constants” show that Pb displays a greater affinity for EPS than Cd. For a given metal, the seven EPS investigated did not display differences regarding the binding strength. This work suggests that a wide range of products of bacterial species isolated from activated sludge exhibit similar binding behaviours for Cd or Pb, which may potentially simplify the modelling of the distribution and the speciation of metals in bacteria-bearing natural systems.

Complete Nucleotide Sequence of the pCTX-M3 Plasmid and Its Involvement in Spread of the Extended-Spectrum β-Lactamase GeneblaCTX-M-3

Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

Isolation of polyhydroxyalkanoates-producing bacteria using a combination of phenotypic and genotypic approach

To develop an efficient approach using a combination of phenotypic and genotypic methods for isolation of environmental bacteria that produce mid-chain-length polyhydroxyalkanoates (mcl-PHAs). A viable-colony staining method using Nile red was used to screen for PHA-producing bacteria followed by a polymerase chain reaction (PCR) screen using primers to amplify the partial nucleic acid sequence of the phaC1 synthase gene for confirmation. Microbes containing lipophilic storage compounds isolated from environmental samples could readily be detected by the colony staining method. They were further examined by Sudan Black staining to highlight the inclusions inside the cells. These isolates were subsequently subjected to PCR analysis. As a result, more than a hundred strains were identified as PHA-positive isolates from this screening approach. These results conclusively demonstrate that environmental bacterial strains able to accumulate the PHAs could readily be obtained by this screening method. We propose a polyphasic approach using a combination of phenotypic and genotypic screening method to rapidly screen and identify bacteria able to produce significant amounts of mcl-PHAs from environment. This approach can be adopted as a rapid screen for micro-organisms able to accumulate PHAs to be used for potential manufacture and other industrial applications.

Étude de l'efficacité d'un produit détergent désinfectant pour sols et surfaces sur les souches bactériennes isolées à partir de l'environnement hospitalier

We studied the sensitivity of the hospital environmental bacterial strains to Surfanios ®, wich is a detergent and disinfectant product for surfaces. This product is used in our establishment since nearly 10 years. This work which relates to 425 bacterial strains proposes to study the possible evolution of the resistance of the bacteria under the pressure of a product biocide used since many years without the change of active ingredient as recommended in pharmaceutical and agroalimentary industry. We developed a micromethod to study the sensitivity of many bacterial strains simultaneously into a 96 wells microplaque. All the Staphylococcus aureus strains ( N = 20) and Staphylococcus with coagulase negative ( N = 308) as 78 Acinetobacter sp. strains remain very sensitive to Surfanios ® according to our study. Target dilution, which is the last dilution not allowing the bacterial growth, is much lower than the manufacturer recommended use dilution. For the Pseudomonas aeruginosa strains tested, target dilution is equal or higher than the dilution of use. Rather than to propose the rotation or the alternation of the products, we recommend the use of Surfanios ® for the bionettoyage of dry surfaces which might be contaminated by Staphylococcus sp. or Acinetobacter sp. and the use of another product or Surfanios ® at a higher concentration, active on Pseudomonas sp. to disinfect wet surfaces which are possible reservoirs for the opportunist bacteria of the hydrous flora.

Antimicrobial-resistant patterns of Escherichia coli and Salmonella strains in the aquatic Lebanese environments

This study is the first to be conducted in Lebanon on the isolation and molecular characterization and the antimicrobial resistance profile of environmental pathogenic bacterial strains. Fifty-seven samples of seawater, sediment, crab, and fresh water were collected during the spring and summer seasons of 2003. The isolation of Escherichia coli and Salmonella using appropriate selective media revealed that 94.7% of the tested samples were contaminated with one or both of the tested bacteria. The polymerase chain reaction (PCR) was then used to identify the species of both bacteria using various sets of primers. Many pathogenic E. coli isolates were detected by PCR out of which two were identified as O157:H7 E. coli. Similarly, the species of many of the Salmonella isolates was molecularly identified. The confirmed isolates of Salmonella and E. coli were then tested using the disk diffusion method for their susceptibility to four different antimicrobials revealing high rates of antimicrobial resistance.

A novel membrane bioreactor enhanced by effective microorganisms for the treatment of domestic wastewater

The activated sludge membrane bioreactor (MBR) has been shown to have some advantages for the processing and reclamation of domestic wastewater. We hypothesized that certain microorganisms, chosen for their abilities to decompose the chemical components of raw sewage, would, when coupled with the MBR, significantly improve the stability and efficiency of this system. We selected environmental bacterial strains which oxidize ammonia and nitrites and produce protease, amylase, and cellulase for the development and testing of a novel biologically enhanced MBR (eMBR). We compared the eMBR with the activated sludge MBR. With the eMBR, the average values of effluent quality were: chemical oxygen demand (COD), 40 mg/l(average efficiency of removal 90.0%); and NH(4) (+)-N, 0.66 mg/l(average efficiency of removal 99.4%). Effluent qualities met the standard and were stable during the entire 90 days of this study. For the activated sludge MBR, the COD removal rate was 91.7%, and the NH(4) (+)-N removal (94.8%) was less than that of the eMBR. Start-up time for the eMBR was only 24-48 h, much shorter than the 7-8 days required to initiate function of the standard MBR. The biomass concentrations of total heterotrophic bacteria and autotrophic bacteria in the eMBR did not fluctuate significantly during the course of the study. Various kinds of microorganisms will establish an ecological balance in the reactor. Compared with the activated sludge MBR, the eMBR not only produced an excellent and stable quality of effluent but also resulted in a shorter time to start-up and significantly improved the efficiency of NH(4) (+)-N removal.

Polynucleobacter Bacteria in the Brackish‐Water Species Euplotes harpa (Ciliata Hypotrichia)

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.

Distribution of transposons Tn5044 and Tn5070 with noncanonical mer operons in environmental bacterial populations

The distribution of unusual mercury resistance transposons, Tn5044 and Tn5070, was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase a subunit. Structural organization of mercury operon Tn5070, containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5040- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes ( stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non- E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.