Sequence Analyses and Phenotypic Characterization Revealed Multidrug Resistant Gene Insertions in the Genomic Region Encompassing Phase 2 Flagellin Encoding fljAB Genes in Monophasic Variant Salmonella enterica Serovar 4,5,12:i:- Isolates From Various Sources in Thailand

Salmonella enterica serovar 4,5,12:i:- (S. 4,5,12:i:-), a monophasic variant of Salmonella Typhimurium (STm) lacking the phase 2 flagellin encoding genes fljAB, has become increasingly prevalent worldwide. The increasing trends in multidrug resistant (MDR) S. 4,5,12:i:- prevalence also pose an important global health threat. Though many reports have characterized phenotypic and genotypic drug resistance of this serovar, few studies have characterized antimicrobial resistance of this serovar in Thailand. In this study, 108 S. 4,5,12:i:- isolates from various sources in Thailand and four international S. 4,5,12:i:- isolates were screened using polymerase chain reaction (PCR) to detect the presence of five target regions which are associated with antimicrobial resistant (AMR) genes, in the genomic region that contained fljAB genes in STm. We determined AMR phenotypes of all isolates by Kirby-Bauer disk diffusion method. Whole genome sequencing (WGS) was performed on 53 representative isolates (based on differences in the pulsed filed gel electrophoresis profiles, the sources of isolate, and the PCR and AMR patterns) to characterize the genetic basis of AMR phenotype and to identify the location of AMR determinants. Based on PCR screening, nine PCR profiles showing distinct deletion patterns of the five target regions have been observed. Approximately 76% of isolates (or 85 of 112 isolates), all of which were Thai isolates, contained five target regions inserted between STM2759 and iroB gene. A total of 21 phenotypic AMR patterns were identified with the predominant AmpST resistant phenotype [i.e., 84% (or 94 of 112) tested positive for resistance to ampicillin, streptomycin, and tetracycline], and 89% (or 100 of 112) were found to be MDR (defined here as resistant to at least three classes of tested antimicrobials). Using WGS data, a total of 24 genotypic AMR determinants belonging to seven different antimicrobial groups were found. AMR determinants (i.e., blaTEM–1, strB-A, sul2, and tetB, conferring resistance to ampicillin, streptomycin, sulfonamides, and tetracycline, respectively) were found to be inserted in a region typically occupied by the phase 2 flagellin encoding genes in STm. These resistant genes were flanked by a number of insertion sequences (IS), and co-localized with mercury tolerance genes. Our findings identify AMR genes, possibly associated with multiple IS26 copies, in the genetic region between STM2759 and iroB genes replacing phase 2 flagellin encoding fljAB genes in Thai S. 4,5,12:i:- isolates.

Prevalence of heterotrophic methylmercury detoxifying bacteria across oceanic regions

Microbial reduction of inorganic divalent mercury (Hg2+) and methylmercury (MeHg) demethylation is performed by the mer operon, specifically by merA and merB genes respectively, but little is known about the mercury tolerance capacity of marine microorganisms and its prevalence in the global ocean. Here, we explored the distribution of these genes in 290 marine heterotrophic bacteria (Alteromonas and Marinobacter spp.) isolated from different oceanographic regions and depths, and assessed their tolerance to diverse concentrations of Hg2+ and MeHg. About 25% of the isolates presented merA and only 8.9% presented both merAB genes, including the strain ISS312 that exhibited the highest tolerance capacity and a degradation efficiency of 98.2% in 24 h. Fragment recruitment analyses of ISS312 genome against microbial metagenomes indicated an extensive distribution across the global bathypelagic ocean. Our findings highlighted that mercury resistance genes are widely distributed in a non-highly polluted environment such as the pelagic marine environment, and that degradation of the neurotoxic MeHg can be performed through the ocean water column by some heterotrophic bacteria at high efficiency with important implications in the biogeochemical cycle of mercury and potentially for the environment and human health.

In Vitro Bioremediation: A Development Process of Cadmium and Mercury Removal by Environmental Biotechnologies of UV-Mutated Escherichia coli K12 and Bacillus subtilis 168

coli K12 and B. subtilis 168 were investigated for their cadmium and mercury tolerance abilities. They were developed by UV mutagenesis technique to increase their tolerances either to cadmium or mercury, and their names then were designated depend on the name and concentration of metals. E. coli K12 Cd3R exhibited bioremediation amount of 6.5 mg Cd/g dry biomass cell. At the same time, its wild-type (E. coli K12 Cd3) was able to remove 5.2 mg Cd/g dry biomass cell in treatment of 17 mg Cd /L within 72 hours of incubation at 37 °C (pH=7) in vitro assays. The results show that E.coli K12 Hg 20 was able to remove 0.050 µg Hg/g dry biomass cell and more removal by its mutant E.coli K12 Hg 20R to 0.060 µg Hg/ g dry biomass cell in the treatment of 0.15 µg Hg /L. On the other hand, B. subtilis168 Cd2 was able to remove the least amount of cadmium (5 mg Cd/ g dry biomass cell) and of mercury (0.045 µg Hg/ g dry biomass cell) under the same conditions were used for E. coli K12. Also, the complete removal of both metals was confirmed by scanning electron microscopy (SEM) showing that the effect of cadmium and mercury on the bacterial mass. Also, the SEM images showed that the removal amounts had relationships in changing the morphology of cells under in vitroassays.

Isolation and characterization of mercury-resistant bacteria from wastewater sources in Egypt

An important mechanism for microbial resistance to mercury is its reduction into elemental mercury (facilitated by the merA gene). Thirty-eight microbial isolates from a variety of wastewater sources in Egypt were collected. Approximately 14 of the 38 isolates exhibited not only a high degree of tolerance to mercury (up to 160 ppm) but also a high degree of resistance to other tested heavy metals (Cu, Co, Ni, and Zn). From these 14, the 10 most resistant isolates were selected for further study and were found to include 9 Gram-negative and 1 Gram-positive bacterial strains. Multi-antibiotic-resistance profiles were detected for 6 out of the 10 selected isolates. All the tested Gram-negative isolates (n = 9) harbored a plasmid-encoded merA gene. The mercury removal effectiveness for the 10 selected isolates ranged between 50% and 99.9%, among which Stenotrophomonas maltophilia ADW10 recorded the highest rate (99.9%; at an initial mercury concentration of 20 ppm). To the best of our knowledge, this is the first study to (i) demonstrate the presence of a multimetal-resistant S. maltophilia bacterium with a high mercury tolerance capacity that would make it a suitable candidate for future bioremediation efforts in heavy-metal-polluted areas in Egypt and (ii) report Pseudomonas otitidis as one of the mercury-resistant bacteria.

Mercury tolerance of Penicillium sp isolated from kefir grains

Inorganic contaminant contamination poses the greatest threat to the environment and human health. It has been recently reported that probiotics protect the body from inorganic contaminant damage by the bioabsorption capacity of its components. Kefir is a beverage obtained through the fermentation metabolized by Lactobacillus and yeasts that aggregate forming the grains of kefir. In order to isolate inorganic contaminant resistant microorganism, the kefir grains were crushed and the supernatant obtained was inoculated in malt-gar medium (2%) and cultured for 120 hours. Occurred formation of halos of growth which were measured every 24 hours. The isolated microorganism was also cultured in liquid medium for five days. The taxonomic analysis revealed to be fungus of the genus Penicillium. The results showed the capacity of mercury bioabsorption by Penicillium sp isolated from kefir. At pH 4.0 this activity did not suffer strong inhibition. Contrary to what was expected there was an increase in biomass when grown in liquid medium which suggests the formation of storage structures of the inorganic contaminant around the cell membrane. These results confirm that kefir contains in its composition microorganisms capable of protecting people who consume it from harmful damages caused by inorganic contaminants.