Chloramphenicol Resistance Gene Research Articles

ChloS-HRM, a novel assay to identify chloramphenicol-susceptible Escherichia coli and Klebsiella pneumoniae in Malawi.

ObjectivesChloramphenicol is a broad-spectrum antimicrobial widely available in sub-Saharan Africa. With susceptibility re-emerging among Enterobacteriaceae in Blantyre, Malawi, we designed and evaluated a new high-resolution melt (HRM) RT-PCR assay, ChloS-HRM, to identify chloramphenicol-susceptible infections in a hospital setting.MethodsSeventy-two previously whole-genome sequenced isolates of Escherichia coli and Klebsiella pneumoniae from the Queen Elizabeth Central Hospital, Malawi, were subjected to determination of chloramphenicol MICs. Primers were designed to detect 18 chloramphenicol resistance genes that produce seven distinct peaks correlating with different gene groups (catA1, catA2, catA3, catB2, catB group 3, cmlA and floR) following HRM analysis. ChloS-HRM results were compared with MIC and WGS results.ResultsChloS-HRM correctly identified 15 of 17 phenotypically susceptible isolates and 54 of 55 resistant isolates, giving an accuracy of 88% in identifying susceptibility and 98% in identifying resistance. WGS identified 16 of 17 susceptible and 54 of 55 resistant isolates, giving an accuracy of 94% in identifying susceptibility and 98% in identifying resistance. The single false-susceptible result had no detectable gene by ChloS-HRM or WGS. Compared with WGS, ChloS-HRM had 100% sensitivity and specificity for catA (catA1–3), cmlA and floR, and 96% specificity for catB; sensitivity could not be estimated due to the lack of catB in the clinical sample collection. The overall agreement between MIC and HRM was 96% and between MIC and WGS it was 97%.ConclusionsChloS-HRM could support antimicrobial stewardship in enabling de-escalation from third-generation cephalosporins by identifying chloramphenicol-susceptible infections. This would be valuable in areas with chloramphenicol-susceptible MDR and XDR Enterobacteriaceae.

Putative antibiotic resistance genes present in extant Bacillus licheniformis and Bacillus paralicheniformis strains are probably intrinsic and part of the ancient resistome.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Poultry as a vector for emerging multidrug resistant Enterococcus spp.: First report of vancomycin (van) and the chloramphenicol–florfenicol (cat-fex-cfr) resistance genes from pigeon and duck faeces

Although commonly regarded as human and animal intestinal tract commensals, Enterococcus spp. have emerged as important nosocomial pathogens due to their intrinsic or acquired resistance to a number of antibiotics. Poultry has been suggested to be a reservoir for antibiotic resistance that may aggravate the problem of transmission of enterococci infections. Between January and December 2016, 106 Enterococcus spp. were isolated from a total of three poultry species. The collection included isolates recovered from chickens (n = 30), ducks (n = 35) and pigeons (n = 41). All enterococci isolates were screened for their ability to form biofilm. The antibiotic susceptibility was determined against 13 antibiotics using the disc diffusion method. The presence of the eight resistance genes, vanA, vanB, vanC, catA, catB, fexA, fexB and cfr was determined by PCR. All 106 isolates were resistant to clindamycin, whereas majority of isolates (>90%) were resistant to erythromycin, oxytetracycline, doxycycline, gentamycin, ciprofloxacin, norfloxacin, and vancomycin. All isolates produced biofilms and were classified as extensive drug-resistant. MARindices for all isolates was determined to be > 0.8, indicating that they have been recovered from high risk contamination sources. The cfr resistance gene was not detected in any of the 106 enterococci isolates, whereas the chloramphenicol resistance genes catA and catB were found in 18.9% (20/106) of the isolates. Interestengly, fexA 11.9% (15/106), fexB 8.7% (11/106), vanA 18.9% (20/106), vanB 25.5% (27/106), and vanC 33% (35/106) genes were also determined in our study. The present study highlights the emergence of a linezolid sensitive-vancomycin resistant enterococci, which lacks the cfr gene reporting also for the first time the detection of van, fex and cat -genes in Enterococcus species recovered from chickens, ducks and pigeons in Egypt suggesting that poultry species could be potential vectors for transmission of multidrug resistant enterococci posing a public health risk.

PEG-mediated, Stable, Nuclear and Chloroplast Transformation of Cyanidioschizon merolae.

The ability to achieve nuclear or chloroplast transformation in plants has been a long standing goal, especially in microalgae research. Over past years there has been only little success, but transient and stable nuclear transformation has been achieved in multiple species. Our newly developed method allows for relatively simple transformation of Cyanidioschizon merolae in both nuclear and chloroplast genome by means of homologous recombination between the genome and a transformation vector. The use of chloramphenicol resistance gene as the selectable marker allows for plate-based efficient selection of mutant colonies. Overall, the method allows the generation of mutant strains within 6 months.

A Method for the Simultaneous Detection of Vibrio cholerae Strains and Drug-Resistant Genes in their Genome by Real-Time PCR

A method was developed for the simultaneous detection of V. cholerae strains and the presence of drug-resistant genes in their genomes by real-time PCR. Resistance to four antibiotics used for cholera treatment (tetracycline, trimethoprim, chloramphenicol and ciprofloxacin) was analyzed. The sensitivity of the panel was 1 × 103 CFU/mL for pure cultures, simulated clinical material, and environmental samples. PCR efficiency was confirmed by the analysis of 60 natural V. cholerae strains isolated from patients and the environment in different years. It was established that all of the studied V. cholerae strains of the O1 serogroup El Tor biovar isolated before 1993 did not contain the tested drug-resistant genes. At the same time, 18 toxigenic clinical strains (90%) imported within 1993–2010 were characterized by multiple drug resistance; their genomes contained trimethoprim resistance genes (dfrA1) and chloramphenicol resistance genes (floR), and 11 strains additionally contained a tetracycline-resistant gene tetR. In addition, nontoxigenic clinical and aqueous V. cholerae strains carrying a ciprofloxacin-resistant gene, the qnrVC (qnrVC1) gene, have been detected in the last few years (Kalmykia, 2011–2013).

Unexpected transcriptome pompTʹ contributes to the increased pathogenicity of a pompT mutant of avian pathogenic Escherichia coli

The lambda red recombination system makes it suitable for screening virulence gene utility in avian pathogenic Escherichia coli (APEC) on account of its wide applicability, simplicity and high efficiency. In APEC E058 (O2 serogroup), there are two copies of the outer membrane protease (ompT) gene, compT encoding cOmpT that is located on the chromosome and pompT encoding pOmpT that is located on a ColV plasmid. However, the relationship between pathogenesis and pompT expression in APEC E058 has yet to be elucidated. Here, we successfully constructed two pompT gene mutants: E058ΔpompT containing a chloramphenicol (cat) resistance gene and E058ΔpompTʹ without the cat gene. By RT-PCR and sequencing analysis, an unexpected transcriptome pompTʹ was detected in mutant strain E058ΔpompTʹ after deletion of the cat gene induced by the lambda red recombination system. Surprisingly, the pathogenicity of mutant E058ΔpompT was significantly attenuated compared to its parental strain in the chicken infection model and HD11 cell model then the pompT gene was knocked out, while the pathogenicity of the other mutant strain E058ΔpompTʹ had no difference. Furthermore, the presence of unexpected transcriptome pompTʹ influenced the bactericidal activity of SPF chicken serum and decreased the transcription level of TLR2 in the heart tissue of chickens. Our study identifies the pompT gene plays an important role in the virulence of APEC E058, and the unexpected transcriptome pompTʹ contributes to the increased pathogenicity of APEC E058 mutants following deletion of the cat gene induced by the lambda red recombination system, which suggests that this system still has some limitations for construction of mutant strains particularly where these are used in development of live vaccine.

A Comprehensive Research on Antibiotic Resistance Genes in Microbiota of Aquatic Animals.

The occurrence of antibiotic resistance genes (ARGs) as emerging contaminants is of continued concern for human health. Antibiotics used in aquaculture have promoted the evolution and spread of ARGs. This study aimed to investigate the occurrence of 37 ARGs conferring resistance to six classes of antibiotics in 94 aquatic animals from five cities in southeast coast of China. The results showed that floR, sulII, sulI, strB, strA, aadA, and tetS were identified as the prominent ARGs with the high detection frequencies ranging from 30.9 to 51.1% in total samples. Then relative expression amount of seven prominent ARGs quantified by qPCR, ranging from 0.003 to 0.065. The tetS was the most abundant ARG among the seven ARGs. Though aadA was the second highest detection frequency of ARGs, it was the lowest expression amount ARG. The occurrences and abundances of ARGs in freshwater aquatic animals were greater than those in marine, reflecting the discrepancy of cultivation pattern between the freshwater and marine aquaculture. Shanghai was considered as the most prevalent site with 16 ARGs, and Ningbo merely contained 9 ARGs without of β-lactam ARGs and quinolone ARGs, showing variations of ARGs with geographical location. Eight kinds of sulfonamides and one chloramphenicol residues were further measured in samples from Shanghai. Interestingly, no target antibiotics were found, but sulfonamides resistance genes (sulI, sulII) and chloramphenicol resistance genes (floR) persisted at aquatic animals in the absence of selection pressure. Our research firstly shows comprehensive information on the ARGs in skin microbiota of aquatic animals, which could provide useful information and a new insight for better understanding on the ARGs dissemination in aquatic animals.

Isolation, Functional Characterization and Transmissibility of p3PS10, a Multidrug Resistance Plasmid of the Fish Pathogen Piscirickettsia salmonis.

Antibiotic resistance is a major public health concern due to its association with the loss of efficacy of antimicrobial therapies. Horizontal transfer events may play a significant role in the dissemination of resistant bacterial phenotypes, being mobilizable plasmids a well-known mechanism. In this study, we aimed to gain insights into the genetics underlying the development of antibiotic resistance by Piscirickettsia salmonis isolates, a bacterial fish pathogen and causative agent of salmonid piscirickettsiosis, and the main target of antibiotics used in Chilean salmon farming. We provide experimental evidence that the plasmid p3PS10, which harbors multidrug resistance genes for chloramphenicol (cat2), tetracyclines [tet(31)], aminoglycosides (sat1 and aadA1), and sulfonamides (sul2), is carried by a group of P. salmonis isolates exhibiting a markedly reduced susceptibility to oxytetracycline in vitro (128–256 μg/mL of minimal inhibitory concentration, MIC). Antibiotic susceptibility analysis extended to those antibiotics showed that MIC of chloramphenicol, streptomycin, and sulfamethoxazole/trimethoprim were high, but the MIC of florfenicol remained at the wild-type level. By means of molecular cloning, we demonstrate that those genes encoding putative resistance markers are indeed functional. Interestingly, mating assays clearly show that p3PS10 is able to be transferred into and replicate in different hosts, thereby conferring phenotypes similar to those found in the original host. According to epidemiological data, this strain is distributed across aquaculture settings in southern Chile and is likely to be responsible for oxytetracycline treatment failures. This work demonstrates that P. salmonis is more versatile than it was thought, capable of horizontally transferring DNA, and probably playing a role as a vector of resistance traits among the seawater bacterial population. However, the low transmission frequency of p3PS10 suggests a negligible chance of resistance markers being spread to human pathogens.

Exotic glycerol dehydrogenase expressing Escherichia coli increases yield of 2,3-butanediol

BackgroundThe thriving of biodiesel industry has led to produce 10% (v/v) crude glycerol, thus creating an overflow problem. Biofuel production is restricted by Escherichia coli due to its toxicity to bacterial cells. Therefore, a platform chemical and fuel additive 2,3-butanediol (2,3-BD) with low toxicity to microbes could be a promising alternative for biofuel production by recombinant E. coli using glycerol as the sole substrate.ResultsA novel expression system of E. coli was developed to express the dhaD gene encoding glycerol dehydrogenase (GDH) to produce value-added metabolic products through aerobic biotransformation of glycerol. The dhaD gene obtained from Klebsiella pneumoniae SRP2 was expressed in E. coli BL21(DE3)pLysS using an E. coli–K. pneumoniae shuttle vector pJET1.2/blunt consisting of chloramphenicol-resistance gene under the control of the T7lac promotor. RT-PCR analysis and dhaD overexpression confirmed that the 2,3-BD synthesis pathway gene was expressed on RNA and protein levels. Therefore, the recombinant E. coli exhibited a 38.9-fold higher enzyme activity (312.57 units/mg protein), yielding 8.97 g/L 2,3-BD, a 2.4-fold increase with respect to the non-recombinant strain.ConclusionsThe engineered strain E. coli BL21(DE3)pLysS/pJET1.2/blunt-dhaD, carrying the 2,3-BD pathway gene dhaD from our newly isolated Klebsiella pneumoniae SRP2 strain, displayed the best ability to synthesize 2,3-BD from low-cost biomass glycerol. The value of expression of an important glycerol metabolism gene dhaD is the highest ever achieved with an engineered E. coli strain. From these results, the first reported dhaD expression system has paved the way for improvement of 2,3-BD production and is efficient for another heterologous gene expression in E. coli.

Phylogenetic Analysis of the Antibiotic Resistance Genes in Salmonella Species in silico.

Antibiotic resistance is an emerging problem in both developed and developing countries. It has been responsible for 700,000 deaths worldwide. Some genotypes of bacteria are sensitive to certain antibiotics than others. Hence by conducting phylogenetic analysis of bacteria and detecting the presence of resistance genes in each genotype, we can select the antibiotic that would be most effective for the bacteria in that certain genotype. A total of forty-five Salmonella species were investigated for the presence of antibiotic resistance genes through in silico PCR (polymerase chain reaction) amplification and PFGE (pulsed-field gel electrophoresis) analysis was conducted to assess the phylogenetic relationship. Total twenty-eight antibiotic resistance genes were selected for screening the isolates and seventeen antibiotic resistance genes among the Salmonella strains were found. Almost all the isolates (n=43) exhibited PCR amplification product for gyrA genes while fluoroquinolone resistance gyrB (66.67%), parC (68.89%) and parE (15.56%) genes were also present. About 15.56% and 11.11% isolates were found to harbor adenylyltransferase gene, aadA1 and aadA2, respectively while phosphotransferase gene was detected in only one isolate. Two isolates expressed both chloramphenicol acetyltransferase genes, cat1 and cat2. Three isolates (6.67%) harbored chloramphenicol resistance gene cmlA gene while two isolates (4.44%) expressed florfenicol resistance gene, floR. Tetracycline resistance gene, tetA was more prevalent (8.89%) than tetG genes (2.22%). Salmonella harbored all three sulfonamide resistance genes while sulII was more prevalent (17.78%). Genotype 2 contained fifteen antibiotic resistance genes while genotype 3 contained only one antibiotic resistance genes. These investigations used a computer aided approach to genotype isolates and assess the difference in antibiotic resistance profile of Salmonella species based on genotype. This data helps to predict antibiotic resistance genes that might be present for an isolate of known genotype and select antibiotic for the treatment of Salmonella infections based on their phylogenetic group.

Antimicrobial Susceptibility and Occurrence of Resistance genes among Salmonella arizonae isolated from Chicken Meat samples in Sokoto Metropolis, Sokoto State, Nigeria

This study was conducted to determine the antimicrobial susceptibility and occurrence of resistance genes among Salmonella Arizonae isolated from chicken meat samples collected in Sokoto metropolis, Sokoto State Nigeria. A total of 400 Chicken’s Meat samples (breast muscle) were collected and examined for the presence of Salmonella organisms. The isolated Salmonella Arizonae were then tested for antimicrobial susceptibility using disc diffusion technique and antimicrobial resistant isolates were then examined for the presence of ampicillin resistance gene blaTEM, chloramphenicol resistance gene floR and tetracycline resistance gene tetA by PCR. Salmonella aizonae were isolated in only 4.5% (18 / 400) of the samples examined. Antimicrobial susceptibility test of the isolates showed susceptibility to nitrofurantoin (94.4%), nalidixic acd (94.4%), oflxacin (77.8%), augmentin (72.2%) and cotrimoxazole (66.7%). However, the isolates were resistant to tetracycline (88.9%), ampicillin (72.2%) and chloramphenicol (66.6%). Only 8 of these isolates were resistant to one or more of the antimicrobial agents tested. Resistance gene analysis of the resistant isolates shows the presence of ampicillin resistance gene, blaTEM in 75% (6 / 8) of the tested isolates. Presence of chloramphenicol resistance gene, floR was also detected in 37.5% (3 / 8) of the isolates. Presence of multiple resistance genes was also detected in the isolates. A combination of two different resistance genes was detected in 62.5% (5 / 8) of the isolates and presence of all the three resistance genes (blaTEM, tetA and floR) was detected in one of the isolates. Antibiotics have been used widely in poultry to treat, prevent infections and also in feeds to promote growth. Such practice has improved poultry performance effectively and economically but has resulted in the increase in the spread of resistant bacterial strains. In our study, a high resistance profile of 88.9% to tetracycline, a popularly use drug in poultry industry and the presence of multiple resistance genes in 62.5% of the isolates was also observed. There is therefore the need for more rational use of antibiotics in animal production and more prudent use in humans.

The rumen microbiome as a reservoir of antimicrobial resistance and pathogenicity genes is directly affected by diet in beef cattle

BackgroundThe emergence and spread of antimicrobial resistance is the most urgent current threat to human and animal health. An improved understanding of the abundance of antimicrobial resistance genes and genes associated with microbial colonisation and pathogenicity in the animal gut will have a major role in reducing the contribution of animal production to this problem. Here, the influence of diet on the ruminal resistome and abundance of pathogenicity genes was assessed in ruminal digesta samples taken from 50 antibiotic-free beef cattle, comprising four cattle breeds receiving two diets containing different proportions of concentrate.ResultsTwo hundred and four genes associated with antimicrobial resistance (AMR), colonisation, communication or pathogenicity functions were identified from 4966 metagenomic genes using KEGG identification. Both the diversity and abundance of these genes were higher in concentrate-fed animals. Chloramphenicol and microcin resistance genes were dominant in samples from forage-fed animals (P < 0.001), while aminoglycoside and streptomycin resistances were enriched in concentrate-fed animals. The concentrate-based diet also increased the relative abundance of Proteobacteria, which includes many animal and zoonotic pathogens. A high ratio of Proteobacteria to (Firmicutes + Bacteroidetes) was confirmed as a good indicator for rumen dysbiosis, with eight cases all from concentrate-fed animals. Finally, network analysis demonstrated that the resistance/pathogenicity genes are potentially useful as biomarkers for health risk assessment of the ruminal microbiome.ConclusionsDiet has important effects on the complement of AMR genes in the rumen microbial community, with potential implications for human and animal health.

Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei.

Numerous lactic acid bacteria (LAB) bacteriophage genomes have been sequenced, while the functional genes are yet to be exploited. In this study, a λ Red-like recombinase operon LCABL_13040-50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome, and its recombination function was confirmed by the replacement of a 167-bp galK fragment with chloramphenicol-resistant gene (cat) in the L. casei BL23 genome. Further functional analysis showed that LCABL_13040 and LCABL_13060 were analogs to the host nuclease inhibitor (Redγ) and 5΄-3΄ exonuclease (Redα/RecE), respectively. After optimization of recombineering conditions, including induction, homology length, recovery time and double-strand DNA substrates quantity, the recombineering efficiency reached ∼2.2×10-7. Subsequently, combining cre-lox technology, the optimal LCABL_13040-50-60 proteins could catalyze markerless deletion of a 167-bp galK fragment and insertion of the gfp gene as well as precision point mutation of rpoB gene in the L. casei BL23 genome, suggesting the LCABL_13040-50-60 operon encoded for three recombineering proteins. Moreover, with the assistance of Redγ, the LCABL_13040-50-60 proteins also showed recombinase activity in six other L. casei strains, L. paracasei OY and L. plantarum WCSF1. All the results demonstrated that the prophage-associated recombinases LCABL_13040-50-60 have great potential to be used for genome editing in LAB.

High-Throughput IgG Reformatting and Expression.

We have recently described a one-step zero-background IgG reformatting method that enables the rapid reformatting of phage-displayed antibody fragments into a single-mammalian cell expression vector for full IgG expression (Chen et al. Nucleic Acids Res 42:e26, 2014). The strategy utilizes our unique positive selection method, referred to as insert-tagged (InTag) positive selection, where a positive selection marker (e.g. chloramphenicol-resistance gene) is cloned together with the antibody inserts into the expression vector. The recombinant clones containing the InTag adaptor are then positively selected without cloning background, thus bypassing the need to plate out cultures and screen colonies. This IgG reformatting method is rapid and can be automated and performed in a high-throughput (HTP) format. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated. We have further optimized the protocol for IgG reformatting since the initial publication of this method (Chen et al. Nucleic Acids Res 42:e26, 2014) and also updated the transient transfection protocol using Expi293F cells, which are described herein.

Comparative gut microbiota and resistome profiling of intensive care patients receiving selective digestive tract decontamination and healthy subjects

BackgroundThe gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed “selective decontamination of the digestive tract” (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes (“the resistome”) of ICU patients during SDD and of healthy subjects.ResultsFrom ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs.The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6′)-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects.ConclusionsThe gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.

Development of the first gene expression system for Salinicoccus strains with potential application in bioremediation of hypersaline wastewaters.

Salinicoccus salsiraiae IM408 (=CGMCC13032) is a novel halophilic bacterium that we isolated from the saline soil of Da Gang Oilfield. It tolerates 60g/l sodium chloride and up to 123g/l (1.5M) sodium acetate and has shown a potential application in bioremediation of wastewater with high salt and high chemical oxygen demand (COD). Two plasmids, pS408-1 and pS408-2, were identified in S. salsiraiae IM408, and the sequences and copy numbers of the plasmids were determined. Based on these plasmids, two shuttle vectors containing a replicon for Escherichia coli, ampicillin, and chloramphenicol resistance genes, as well as the replicon from pS408-1 or pS408-2, were constructed and named as pTCS101 and pTCS201, respectively. A suitable host strain, named S. salsiraiae PE01, was also developed from the wild-type by plasmid elimination. Using the plasmid pTCS101 as an expression vector, L-lactate dehydrogenase from Staphylococcus aureus was expressed successfully in S. salsiraiae PE01. This is the first gene expression system for the Salinicoccus genus. It has provided the potential for expression of desired proteins or for establishment of desired pathways in Salinicoccus strains, which would make these halophiles more advantageous in future biotechnological applications.

The behavior of antibiotic resistance genes and arsenic influenced by biochar during different manure composting.

The effect of two different biochar types, rice straw biochar (RSB) and mushroom biochar (MB), on chicken manure composting was previously examined by monitoring the fate of antibiotic resistance genes (ARGs) and arsenic. The behavior of ARGs and arsenic in other kinds of manure composting with the same biochar types had not been examined. In this study, we added either RSB or MB to pig and duck manure composts to study the behavior of ARGs (tet genes, sul genes, and chloramphenicol resistance genes) and arsenic under the same experimental condition. The results showed that the average removal values of selected ARGs were respectively 2.56 and 2.09 log units in duck and pig manure compost without the addition of biochar. The effect of biochar addition on the average removal value of ARGs depended on the type of biochar and manure. For instance, in pig manure compost, MB addition increased the average removal value of ARGs, while RSB addition decreased. And both biochar additions had a negative influence on the average removal value of ARGs in duck manure compost. Analytical results also demonstrated that MB addition reduced total arsenic and the percentage of bioavailable arsenic more than RSB.

Effect of bio-electrochemical system on the fate and proliferation of chloramphenicol resistance genes during the treatment of chloramphenicol wastewater

Bioelectrochemical systems can effectively degrade antibiotics, but there is the need to better understand the fate of antibiotic resistance bacteria and antibiotic resistance genes during the bioelectrochemical degradation of antibiotics. In this study, a BES was developed as a platform to investigate the fate of chloramphenicol resistance bacteria (CRB) and the expression of chloramphenicol resistance genes (CRGs) under different operating conditions during chloramphenicol biodegradation. The results indicated that chloramphenicol was effectively removed and chloramphenicol removal efficiency could be improved under less chloramphenicol concentration and more negative cathode potential. Higher chloramphenicol concentration enhanced the enrichment of CRB and expression of CRGs. Furthermore, the abundances of CRB were enhanced under more negative cathode potential, the expression of CRGs under less negative cathode potential were induced. However, both the enrichment of CRB and expression of CRGs could be moderated under a medium cathode potential. This result could provide the scientific reference for research about the fate of antibiotic resistance genes in bioelectrochemical systems.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis -Acting Element That Is Essential for Nucleocapsid Assembly

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83-encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis-acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis-acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis-acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process.IMPORTANCE Virus nucleocapsid assembly usually requires specific cis-acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis-acting elements in the viral genome.