Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics

The effects of antibiotics on environment-originated nonpathogenic Acinetobacter species have been poorly explored. To understand the antibiotic-resistance mechanisms that function in nonpathogenic Acinetobacter species, we used an RNA-sequencing (RNA-seq) technique to perform global gene-expression profiling of soil-borne Acinetobacter oleivorans DR1 after exposing the bacteria to 4 classes of antibiotics (ampicillin, Amp; kanamycin, Km; tetracycline, Tc; norfloxacin, Nor). Interestingly, the well-known two global regulators, the soxR and the rpoE genes are present among 41 commonly upregulated genes under all 4 antibiotic-treatment conditions. We speculate that these common genes are essential for antibiotic resistance in DR1. Treatment with the 4 antibiotics produced diverse physiological and phenotypic changes. Km treatment induced the most dramatic phenotypic changes. Examination of mutation frequency and DNA-repair capability demonstrated the induction of the SOS response in Acinetobacter especially under Nor treatment. Based on the RNA-seq analysis, the glyoxylate-bypass genes of the citrate cycle were specifically upregulated under Amp treatment. We also identified newly recognized non-coding small RNAs of the DR1 strain, which were also confirmed by Northern blot analysis. These results reveal that treatment with antibiotics of distinct classes differentially affected the gene expression and physiology of DR1 cells. This study expands our understanding of the molecular mechanisms of antibiotic-stress response of environment-originated bacteria and provides a basis for future investigations.     

Identification and characterization of genes regulated by AqsR, a LuxR-type regulator in Acinetobacter oleivorans DR1

The complete genome of Acinetobacter oleivorans DR1 contains AqsR and AqsI genes, which are LuxR and LuxI homolog, respectively. In a previous study, we demonstrated that quorum sensing (QS) signals play an important role in biofilm formation and hexadecane biodegradation. However, the regulation of genes controlled by the QS system in DR1 remains unexplored. We constructed an aqsR mutant and performed RNA sequencing analysis to understand the QS system. A total of 353 genes were differentially expressed during the stationary phase of wild-type cells compared to that of the aqsR mutant. AqsR appears to be an exceptionally important regulator because knockout of aqsR affected global gene expression. Genes involved in posttranslational modification, chaperones, cell wall structure, secondary metabolites biosynthesis, and stress defense were highly upregulated only in the wild type. Among upregulated genes, both the AOLE_03905 (putative surface adhesion protein) and the AOLE_11355 (L-asparaginase) genes have putative LuxR binding sites at their promoter regions. Soluble AqsR proteins were successfully purified in Escherichia coli harboring both aqsR and aqsI. Comparison of QS signals in an AqsI–AqsR co-overexpression strain with N-acyl homoserine lactone standards showed that the cognate N-acyl homoserine lactone binding to AqsR might be 3OH C12HSL. Our electrophoretic mobility shift assays with purified AqsR revealed direct binding of AqsR to those promoter regions. Our data showed that AqsR functions as an important regulator and is associated with several phenotypes, such as hexadecane utilization, biofilm formation, and sensitivity to cumene hydroperoxide.     

Co-Transduction of Plasmids Mediating Resistance to Tetracycline and Chloramphenicol in Staphylococcus aureus

Independent plasmids mediating resistance either to tetracycline (Tc) or chloramphenicol (Cm) were transduced successively into Staphylococcus aureus strain 8325. From this doubly resistant donor strain, Tc was co-transduced with a frequency of 40 to 50% when Cm was selected. Co-transduction of Cm was 5 to 10% with Tc selection. Plasmid elimination was infrequent and restricted to the Cm plasmid. A variant, doubly resistant strain gave 100% co-transduction of Tc and Cm and a high rate of joint elimination of both plasmid markers. Co-transduction of the plasmids from recombination-deficient donor strains was much reduced if the plasmids had been introduced separately into the donor strain, but occurred at the normal high rate if they had been introduced jointly. The plasmids were co-transformed at relatively low rates with closed circular deoxyribonucleic acid (DNA) from doubly resistant donors, but not with DNA from a mixed lysate of singly resistant strains. Our evidence favored a hypothesis of recombination-dependent, reversible linkage between the two plasmids as the basis for their co-transduction. Examination of plasmid DNA from the doubly resistant strains by ultracentrifugal and electron microscopic methods did not disclose any physical differences between singly and doubly resistant strains that might account for the observed co-transduction.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: IV. Adaptation of Activated Sludge to Utilization of Aromatic Compounds 1

An activated sludge from a sewage treatment plant and a laboratory activated sludge developed on an artificial waste were compared for their ability to utilize 11 aromatic compounds. There were several significant differences between them. The laboratory sludge contained higher numbers of organisms and metabolized the aromatics to a greater extent. Laboratory activated sludges acclimated to utilization of the aromatics differed from each other in population structure and the pattern of oxygen consumption with aromatic substrates. The oxidative patterns of uncontrolled mixed populations were unreliable for investigating metabolic pathways. Extracts of the various sludges elevated the plate counts of the sludges.     

海口市污水处理厂活性污泥中细菌的分离鉴定及耐药性分析

为了解海口市白沙门污水处理厂活性污泥中细菌抗生素耐性情况,采用平板分离技术分离、纯化细菌,并通过BIOLOG微生物鉴定系统对筛选到的细菌进行鉴定,同时采用Kirby-Bauer纸片琼脂扩散法进行药敏试验并进行抗生素耐性分析。本研究共分离到18株细菌,分属8个属,14个种,其中G+和G-均为9株。抗生素药敏性试验结果表明,所有菌株均耐药,菌株单重耐药率、双重耐药性及多重耐药性分别为50%、38.9%、和11.1%。菌株对9种常用抗生素:头孢他啶、环丙沙星、庆大霉素、链霉素、氨苄西林、红霉素、氯霉素、四环素、卡那霉素的耐药率分别为61.1%、0%、5.6%、16.7%、50%、16.7%、11.1%、0%、5.6%。综上所述,白沙门污水处理厂活性污泥中的细菌耐药性比较严重,存在潜在的环境生态和人畜健康风险。本研究揭示了当前白沙门污水处理厂活性污泥中细菌对常见抗生素耐药的严重现状,为建议污水处理厂加强出水及污泥中抗生素耐药性及耐药基因的检测并评估其生态影响提供基础,避免出水及污泥中的抗性菌和耐药基因可能带来的风险问题。     

Mutated Cadherin Alleles from a Field Population of Helicoverpa armigera Confer Resistance to Bacillus thuringiensis Toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r₁) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r₂ and r₃) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r₂ and r₃ alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.     

Microbial Community Dynamics and Activity Link to Indigo Production from Indole in Bioaugmented Activated Sludge Systems

Biosynthesis of the popular dyestuff indigo from indole has been comprehensively studied using pure cultures, but less has been done to characterize the indigo production by microbial communities. In our previous studies, a wild strain Comamonas sp. MQ was isolated from activated sludge and the recombinant Escherichia coli _nagAc carrying the naphthalene dioxygenase gene (nag) from strain MQ was constructed, both of which were capable of producing indigo from indole. Herein, three activated sludge systems, G1 (non-augmented control), G2 (augmented with Comamonas sp. MQ), and G3 (augmented with recombinant E. coli _nagAc), were constructed to investigate indigo production. After 132-day operation, G3 produced the highest yields of indigo (99.5 ± 3.0 mg/l), followed by G2 (27.3 ± 1.3 mg/l) and G1 (19.2 ± 1.2 mg/l). The microbial community dynamics and activities associated with indigo production were analyzed by Illumina Miseq sequencing of 16S rRNA gene amplicons. The inoculated strain MQ survived for at least 30 days, whereas E. coli _nagAc was undetectable shortly after inoculation. Quantitative real-time PCR analysis suggested the abundance of naphthalene dioxygenase gene (nagAc) from both inoculated strains was strongly correlated with indigo yields in early stages (0–30 days) (P < 0.001) but not in later stages (30–132 days) (P > 0.10) of operation. Based on detrended correspondence analysis (DCA) and dissimilarity test results, the communities underwent a noticeable shift during the operation. Among the four major genera (> 1% on average), the commonly reported indigo-producing populations Comamonas and Pseudomonas showed no positive relationship with indigo yields (P > 0.05) based on Pearson correlation test, while Alcaligenes and Aquamicrobium, rarely reported for indigo production, were positively correlated with indigo yields (P < 0.05). This study should provide new insights into our understanding of indigo bio-production by microbial communities.     

Distribution of Tetracycline and Streptomycin Resistance Genes and Class 1 Integrons in Enterobacteriaceae Isolated from Dairy and Nondairy Farm Soils

The prevalence of selected tetracycline and streptomycin resistance genes and class 1 integrons in Enterobacteriaceae (n = 80) isolated from dairy farm soil and nondairy soils was evaluated. Among 56 bacteria isolated from dairy farm soils, 36 (64.3%) were resistant to tetracycline, and 17 (30.4%) were resistant to streptomycin. Lower frequencies of tetracycline (9 of 24 or 37.5%) and streptomycin (1 of 24 or 4.2%) resistance were observed in bacteria isolated from nondairy soils. Bacteria (n = 56) isolated from dairy farm soil had a higher frequency of tetracycline resistance genes including tetM (28.6%), tetA (21.4%), tetW (8.9%), tetB (5.4%), tetS (5.4%), tetG (3.6%), and tetO (1.8%). Among 24 bacteria isolated from nondairy soils, four isolates carried tetM, tetO, tetS, and tetW in different combinations; whereas tetA, tetB, and tetG were not detected. Similarly, a higher prevalence of streptomycin resistance genes including strA (12.5%), strB (12.5%), ant(3″) (12.5), aph(6)-1c (12.5%), aph(3″) (10.8%), and addA (5.4%) was detected in bacteria isolated from dairy farm soils than in nondairy soils. None of the nondairy soil isolates carried aadA gene. Other tetracycline (tetC, tetD, tetE, tetK, tetL, tetQ, and tetT) and streptomycin (aph(6)-1c and ant(6)) resistance genes were not detected in both dairy and nondairy soil isolates. A higher distribution of multiple resistance genes was observed in bacteria isolated from dairy farm soil than in nondairy soil. Among 36 tetracycline- and 17 streptomycin-resistant isolates from dairy farm soils, 11 (30.6%) and 9 (52.9%) isolates carried multiple resistance genes encoding resistance to tetracycline and streptomycin, respectively, which was higher than in bacteria isolated from nondairy soils. One strain each of Citrobacter freundii and C. youngae isolated from dairy farm soils carried class 1 integrons with different inserted gene cassettes. Results of this small study suggest that the presence of multiple resistance genes and class 1 integrons in Enterobacteriaceae in dairy farm soil may act as a reservoir of antimicrobial resistance genes and could play a role in the dissemination of these antimicrobial resistance genes to other commensal and indigenous microbial communities in soil. However, additional longer-term studies conducted in more locations are needed to validate this hypothesis.     

Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.     

Phenotypic and Physiological Changes in <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. Strain DR1 with Exogenous Plasmid

The genus Acinetobacter has been recognized to take up exogenous DNA from the environment. In this study, we conducted natural transformation with a novel diesel-degrading Acinetobacter sp. strain, designated strain DR1, using the broad host range plasmid pRK415. Many factors, including temperature, quantities of DNA, and aeration have proven critically important for efficient natural transformation. Interestingly, the Acinetobacter sp. strain DR1 (pRK415) differed both phenotypically and physiologically from the wild-type strain in several regards, including motility, biofilm formation ability, and responses to oxidative stress: the transformed cells were rendered more sensitive to hydrogen peroxide and cumene hydroperoxide, and their motilities and biofilm formation activity were also attenuated. Our data demonstrated that caution should be exercised when conducting genetic manipulation with plasmids, due to the possibility that phenotypic and physiological changes in the host might occur along with the uptake of plasmids.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 Resistance to Zinc Pyrithione: Phenotypic Changes Suggest the Involvement of Efflux Pumps

The aim of this study is to investigate the involvement of an efflux pump in the development of Pseudomonas aeruginosa resistance to zinc pyrithione (ZnPT). In the presence of efflux inhibitor carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the minimum inhibitory concentration of ZnPT for P. aeruginosa resistant cells is reduced significantly (p < 0.05). In addition, the concentration of ZnPT excluded by the resistant bacteria was reduced significantly (p < 0.01). However, the above reductions did not reach the levels measured for P. aeruginosa PAO1 sensitive strain. Furthermore, such changes in P. aeruginosa resistant cells were correlated with the overexpression of outer membrane proteins, reduced sensitivity toward imipenem (p < 0.01) and increased sensitivity toward sulphatriad and chloramphenicol (p < 0.05). In a continuation to a previous study, we conclude that P. aeruginosa resistance to ZnPT is multifactorial and involves induced efflux systems. Suzanne Abdel Malek is currently on leave from Petra University, and a member at the Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.     

Glycans Flanking the Hypervariable Connecting Peptide between the A and B Strands of the V1/V2 Domain of HIV-1 gp120 Confer Resistance to Antibodies That Neutralize CRF01_AE Viruses

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.