Interplays of mutations in waaA, cmk, and ail contribute to phage resistance in Yersinia pestis.

Plague caused by Yersinia pestis remains a public health threat worldwide. Because multidrug-resistant Y. pestis strains have been found in both humans and animals, phage therapy has attracted increasing attention as an alternative strategy against plague. However, phage resistance is a potential drawback of phage therapies, and the mechanism of phage resistance in Y. pestis is yet to be investigated. In this study, we obtained a bacteriophage-resistant strain of Y. pestis (S56) by continuously challenging Y. pestis 614F with the bacteriophage Yep-phi. Genome analysis identified three mutations in strain S56: waaA* (9-bp in-frame deletion 249GTCATCGTG257), cmk* (10-bp frameshift deletion 15CCGGTGATAA24), and ail* (1-bp frameshift deletion A538). WaaA (3-deoxy-D-manno-octulosonic acid transferase) is a key enzyme in lipopolysaccharide biosynthesis. The waaA* mutation leads to decreased phage adsorption because of the failure to synthesize the lipopolysaccharide core. The mutation in cmk (encoding cytidine monophosphate kinase) increased phage resistance, independent of phage adsorption, and caused in vitro growth defects in Y. pestis. The mutation in ail inhibited phage adsorption while restoring the growth of the waaA null mutant and accelerating the growth of the cmk null mutant. Our results confirmed that mutations in the WaaA-Cmk-Ail cascade in Y. pestis contribute to resistance against bacteriophage. Our findings help in understanding the interactions between Y. pestis and its phages.

A potential tool for predicting epidemic trends and outbreaks of scrub typhus based on Internet search big data analysis in Yunnan Province, China.

Introduction: Scrub typhus, caused by Orientia tsutsugamushi, is a neglected tropical disease. The southern part of China is considered an important epidemic and conserved area of scrub typhus. Although a surveillance system has been established, the surveillance of scrub typhus is typically delayed or incomplete and cannot predict trends in morbidity. Internet search data intuitively expose the public's attention to certain diseases when used in the public health area, thus reflecting the prevalence of the diseases. Methods: In this study, based on the Internet search big data and historical scrub typhus incidence data in Yunnan Province of China, the autoregressive integrated moving average (ARIMA) model and ARIMA with external variables (ARIMAX) model were constructed and compared to predict the scrub typhus incidence. Results: The results showed that the ARIMAX model produced a better outcome than the ARIMA model evaluated by various indexes and comparisons with the actual data. Conclusions: The study demonstrates that Internet search big data can enhance the traditional surveillance system in monitoring and predicting the prevalence of scrub typhus and provides a potential tool for monitoring epidemic trends of scrub typhus and early warning of its outbreaks.

High prevalence of Rickettsia spp. in ticks from wild hedgehogs rather than domestic bovine in Jiangsu province, Eastern China.

Background: Spotted fever group Rickettsia (SFGR), containing various pathogenic Rickettsia spp., poses remarkable negative influences to public health by causing various severe or mild diseases. Information regarding prevalence of SFGR in ticks in Jiangsu province, Eastern China, is still limited and needs urgent investigations. Methods: Hedgehog- and bovine-attached ticks were collected from Jiangsu province, Eastern China. DNA of individual ticks was extracted for nested polymerase chain reaction amplifications targeting gltA, 16S ribosomal RNA (rrs), ompA, ompB, and sca4 genes following with sequencing. SFGR-specific IgG antibodies in sera of local donators were evaluated using ELISA. Results: Overall, 144 (83.2%) of the 173 ticks from hedgehogs and 2 (1.2%) of the 168 ticks from bovine were positive for one of the three identified Rickettsia spp., with significant difference between the two groups (P = 3.6e-52). Candidatus Rickettsia principis (9; 5.2%) and R. heilongjiangensis (135; 78.0%) were detected in Haemaphysalis flava rather than in H. longicornis ticks from hedgehogs. R. heilongjiangensis (1; 0.6%) and Candidatus R. jingxinensis (or Candidatus R. longicornii) (1; 0.6%) were identified in H. longicornis and Rhipicephalus microplus ticks from bovine, respectively. Phylogenetic analysis indicated Candidatus R. jingxinensis belonged to R. japonica subgroup, whereas Candidatus R. principis belonged to a novel subgroup. Higher serological prevalence of spotted fever and SFGR-specific IgG antibody level in humans were observed around the investigated area than in urban areas, without significant difference. Conclusion: Candidatus R. principis and Candidatus R. jingxinensis were identified in Jiangsu province, Eastern China, and fully genetically characterized for the first time. The higher prevalence of SFGR in hedgehog-attached ticks as well as the higher SFGR-specific IgG antibody level and seropositive rate in humans around the investigated area suggested that more attention should be paid to SFGR. This pathogen is usually transmitted or harbored by wild animals and ticks. This study provides important epidemiological data for both physicians and public health officers in developing early prevention and control strategies against potential Rickettsia infections and in the preparation of suitable testing and treatment needs for rickettsiosis in the endemic areas.

Wild Hedgehogs and Their Parasitic Ticks Coinfected with Multiple Tick-Borne Pathogens in Jiangsu Province, Eastern China.

The increasing awareness of emerging tickborne pathogens (TBPs) has inspired much research. In the present study, the coinfections of TBPs both in ticks and their wild hedgehog hosts in Jiangsu province, Eastern China were determined by metagenome next-generation sequencing and nested PCR. As a result, Rickettsia japonica (81.1%), novel Rickettsia sp. SFGR-1 (5.1%), Anaplasma bovis (12%), A. platys (6.3%), novel Ehrlichia spp. Ehr-1 (16%) and Ehr-2 (0.6%), E. ewingii-like strain (0.6%), Coxiella burnetii (10.9%), and a novel Coxiella-like endosymbiont (CLE) strain (61.1%) were detected in Haemaphysalis flava ticks. A. bovis (43.8%), Ehrlichia sp. Ehr-1 (83.3%), and C. burnetii (80%) were detected in Erinaceus amurensis hedgehogs. Coinfection rates with various TBPs were 71.5% and 83.3% in ticks and hedgehogs, respectively, both with double-pathogen/endosymbiont coinfection rates over 50%. We found the following. (i) Er. amurensis hedgehogs seem to contribute to the natural cycles of R. japonica, A. bovis, Ehrlichia sp., and C. burnetii and may be reservoirs of them except for R. japonica, and A. bovis is proved to infect hedgehogs for the first time. (ii) H. flava is proved to harbor various TBPs as a reservoir host, including CLE identified for the first time, which could inhibit coinfection of C. burnetii while promoting that of Rickettsia spp. in H. flava. (iii) Four novel TBP species were identified. This study provides useful epidemiological information crucial for assessing the potential infection risks to humans, thus benefiting the development of strategies to prevent and control tick-borne diseases. IMPORTANCE In the present study, we found the following. (i) Er. amurensis hedgehogs seem to contribute to the natural cycles of R. japonica, A. bovis, Ehrlichia sp., and C. burnetii and may be reservoirs of them except for R. japonica, and A. bovis is proved to infect hedgehogs for the first time. (ii) H. flava is proved to harbor various tickborne pathogens (TBPs) as a reservoir host, including Coxiella-like endosymbiont (CLE) identified for the first time, which could inhibit coinfection of C. burnetii while promoting that of Rickettsia spp. in H. flava. (iii) Four novel TBP species were identified. This study provides useful epidemiological information on TBPs harbored and transmitted by ticks and their hosts, for assessing the potential infection risks to humans, thus benefiting the developing strategies for tick-borne diseases prevention and control.

Co-existence of Multiple Anaplasma Species and Variants in Ticks Feeding on Hedgehogs or Cattle Poses Potential Threats of Anaplasmosis to Humans and Livestock in Eastern China.

Background: Anaplasma spp., causative agents of anaplasmosis, pose significant a threat to public health and economic losses in livestock farming. Co-infections/co-existence of various Anaplasma spp. may facilitate pathogen interactions and the emergence of novel variants, represent potential dangers to public health and economic losses from livestock farming, and raise challenges of detection and diagnosis. The information regarding co-infection/co-existence of Anaplasma in their vector ticks and wild animals is limited and needs urgent investigation. Methods: Wild hedgehogs and ticks from hedgehogs and cattle were collected from Jiangsu province, Eastern China, and DNA was extracted from hedgehog organs and tick homogenates. Various genera of species-specific polymerase chain reaction (PCR) or nested PCR amplifications targeting 16S ribosomal RNA (rrs), msp4, or groEL gene coupled with sequencing were conducted to identify Anaplasma spp. Results: Anaplasma phagocytophilum (1, 0.6%), A. marginale (2, 1.2%), A. platys variants xyn10pt-1 (13, 7.7%), xyn21pt-2 (3, 1.8%), and xyn3pt-3 (3, 1.8%), A. bovis variant cwp72bo-1 (12, 7.1%), and a novel Candidatus Cryptoplasma sp. (1, 0.6%) were identified in 168 Haemaphysalis longicornis ticks from cattle. A. platys variant xyn10pt-1 (20, 11.4%) and A. bovis variants cwp72bo-1 (12, 6.9%) and cwp55-36bo-2 (1, 0.6%) were detected in 173 H. flava ticks from hedgehogs. However, only A. bovis variant cwp72bo-1 (15, 46.7%) was identified in 32 Erinaceus amurensis hedgehogs. Various co-existence combinations were found only in ticks. Conclusion: The co-existence of various Anaplasma spp. and variants in H. flava and H. longicornis was detected for the first time in the world. The high infection rate of A. bovis in hedgehogs and its moderate infection rate in their parasitic ticks suggest that Er. amurensis hedgehog could be an important reservoir of A. bovis, rather than A. platys. Horizontal transmission of Anaplasma spp. may exist among different tick species via their shared hosts in the investigated area. This study provided epidemiological data that could be crucial for strategy development for early warning, prevention, and control of potential Anaplasma infections.

Attenuation of Yersinia pestis fyuA Mutants Caused by Iron Uptake Inhibition and Decreased Survivability in Macrophages.

Yersinia pestis is the etiological agent of plague, a deadly infectious disease that has caused millions of deaths throughout history. Obtaining iron from the host is very important for bacterial pathogenicity. Y. pestis possesses many iron uptake systems. Yersiniabactin (Ybt) plays a major role in iron uptake in vivo and in vitro, and in virulence toward mice as well. FyuA, a ß-barrel TonB-dependent outer membrane protein, serves as the receptor for Ybt. In this study, we examined the role of the fyuA gene in Y. pestis virulence using different challenging ways and explored the underlying mechanisms. The BALB/c mouse infection assay showed that the virulence of the mutant strains (ΔfyuA and ΔfyuAGCAdel) was lower when compared with that of the wild-type (WT) strain 201. Furthermore, the attenuation of virulence of the mutant strains via subcutaneous and intraperitoneal challenges was far greater than that via intravenous injection. Iron supplementation restored lethality during subcutaneous challenge with the two mutants. Thus, we speculated that the attenuated virulence of the mutant strains toward the mice may be caused by dysfunctional iron uptake. Moreover, ΔfyuA and ΔfyuAGCAdel strains exhibited lower survival rates in murine RAW264.7 macrophages, which might be another reason for the attenuation. We further explored the transcriptomic differences between the WT and mutant strains at different temperatures and found that the expressions of genes related to Ybt synthesis and its regulation were significantly downregulated in the mutant strains. This finding indicates that fyuA might exert a regulatory effect on Ybt. Additionally, the expressions of the components of the type III secretion system were unexpectedly upregulated in the mutants, which is inconsistent with the conventional view that the upregulation of the virulence genes enhances the virulence of the pathogens.

A multi-epitopes tandem antigen for five types of human adenoviruses and its application in development of multivalent IgM immunochromatographic strip test.

In China, human adenovirus serotype 3 (HAdV-3), HAdV-7, HAdV-11, HAdV-14, and HAdV-55 are the main prevalent serotypes causing severe acute respiratory diseases and even deaths. To develop multivalent vaccine and diagnostic reagent, a multi-epitopes tandem antigen (META) was designed. Recombinant META was prepared and its humoral immunogenicity, inducing neutralization antibody ability, antigenicity, and reactogenicity were evaluated. A multivalent immunochromatographic strip constructed using the rMETA was evaluated for its sensitivity and specificity in detecting specific IgM antibodies. As a result, the rMETA induced high titers of specific IgG antibodies, with limited abilities of neutralizing multiple HAdVs. It performed both strong antigenicity and reactogenicity. The multivalent immunochromatographic strip recognized specific IgM antibodies against all the 5 types with sensitivities of 87.5% to 95.3%. It performed high specificity of 97.8%. The present study provides both novel idea for developing multivalent vaccine and reagent for point-of-care detection of multiple types of HAdVs.

Polygenic plague resistance in the great gerbil uncovered by population sequencing.

Pathogens can elicit high selective pressure on hosts, potentially altering genetic diversity over short evolutionary timescales. Intraspecific variation in immune response is observable as variable survivability from specific infections. The great gerbil (Rhombomys opimus) is a rodent plague host with a heterogenic but highly resistant phenotype. Here, we investigate the genomic basis for plague-resistant phenotypes by exposing wild-caught great gerbils to plague (Yersinia pestis). Whole genome sequencing of 10 survivors and 10 moribund individuals revealed a subset of genomic regions showing elevated differentiation. Gene ontology analysis of candidate genes in these regions demonstrated enrichment of genes directly involved in immune functions, cellular metabolism and the regulation of apoptosis as well as pathways involved in transcription, translation, and gene regulation. Transcriptomic analysis revealed that the early activated great gerbil immune response to plague consisted of classical components of the innate immune system. Our approach combining challenge experiments with transcriptomics and population level sequencing, provides new insight into the genetic background of plague-resistance and confirms its complex nature, most likely involving multiple genes and pathways of both the immune system and regulation of basic cellular functions.

Development of Rapid and Visual Nucleic Acid Detection Methods towards Four Serotypes of Human Adenovirus Species B Based on RPA-LF Test.

OBJECTIVES: Human adenoviruses (HAdV) are classified as 7 HAdV species, and some serotypes in species B like HAdV 3, HAdV 7, HAdV 21, and HAdV 55 caused severe symptoms, even fatalities. Patients may be misdiagnosed and inadequately treated without reliable and practical methods for HAdV serotyping. Developing rapid, sensitive, and specific diagnostic methods for HAdV is critical. METHODS: Detection methods were established based on a recombinase polymerase amplification (RPA) assay and lateral flow (LF) test. Specific target sequence was screened, targeting which, primers and probes were designed, synthesized, and screened for establishing assay with high amplification efficiency. Primer or probe concentrations and amplification time were optimized. Detection limit, sensitivity, and specificity were evaluated. Results and Conclusions. Simple, sensitive, and specific RPA-LF methods for detection of four serotypes of HAdV together or separately were established, which had detection limits of 10 to 280 copies/reaction comparable to real-time PCR without recognizing other pathogens. The sensitivity and specificity were >92% and >98%, respectively, evaluated by limited clinical samples. The detection can be completed in 25 min without requirement of any instrument except a constant temperature equipment, showing superior detection performance and promising for a wide use in the field and resource-limited area.

Genetic diversity and population structure of Aedes aegypti after massive vector control for dengue fever prevention in Yunnan border areas.

Dengue fever is a mosquito-borne disease caused by the dengue virus. Aedes aegypti (Ae. Aegypti) is considered the primary vector of Dengue virus transmission in Yunnan Province, China. With increased urbanization, Ae. aegypti populations have significantly increased over the last 20 years. Despite all the efforts that were made for controlling the virus transmission, especially on border areas between Yunnan and Laos, Vietnam, and Myanmar (dengue-endemic areas), the epidemic has not yet been eradicated. Thus, further understanding of the genetic diversity, population structure, and invasive strategies of Ae. aegypti populations in the border areas was vital to uncover the vector invasion and distribution dynamic, and essential for controlling the infection. In this study, we analyzed genetic diversity and population structure of eight adult Ae. Aegypti populations collected along the border areas of Yunnan Province in 2017 and 2018. Nine nuclear microsatellite loci and mitochondrial DNA (mtDNA) sequences were used to achieve a better understanding of the genetic diversity and population structure. One hundred and fourteen alleles were found in total. The polymorphic information content value, together with the expected heterozygosity (He) and observed heterozygosity (Ho) values showed high genetic diversity in all mosquito populations. The clustering analysis based on Bayesian algorithm, the UPGMA and DAPC analysis revealed that all the eight Ae. aegypti populations can be divided into three genetic groups. Based on the mtDNA results, all Ae. aegypti individuals were divided into 11 haplotypes. The Ae. aegypti populations in the border areas of Yunnan Province presented with high genetic diversity, which might be ascribed to the continuous incursion of Ae. aegypti.

A COVID-19 case report from asymptomatic contact: implication for contact isolation and incubation management.

BACKGROUND: As of 2 March, 2020, at least 80 151 coronavirus disease 2019 (COVID-19) cases were reported in China. Most of the patients had a history of visiting Hubei Province or contacting with people who had ever stayed in or passed by Hubei Province or were exposed to symptoms. Some patients got infected through only asymptomatic contact. This study aimed to report the epidemic features and lab identification of a patient confirmed with COVID-19 infection through only asymptomatic contact. CASE PRESENTATION: A 44-year-old man, who lived in Nanchang, Jiangxi Province, China until 6 March 2020, suffered from cough on 27 January 2020. Fever symptoms appeared on 28 January, with a maximum temperature of 38.8 °C, accompanied by cough, sore throat, headache, fatigue, muscle ache, joint ache, and other symptoms. The symptoms continued until he was hospitalized on 30 January. Coronavirus conventional polymerase chain reaction assay was positive for the throat swab sample. The patient, along with his wife and son, drove from Nanchang to back to Honghu City, Hubei Province, on 23 January 2020. After staying with his parents and brother's family for 3 days, the patient drove back to Nanchang and arrived on 25 January. On the way back home, they stopped by Tongshan service area, Hubei Province, without any close contact with other people. After arriving home in Nanchang City, Jiangxi Province, none of them left their residence. In addition, his parents stayed at home for 20 days with his younger brother's family before they got back. His younger brother and one of his brother's children visited Wuhan on 5 January and came home on 6 January 2020. CONCLUSIONS: This report suggested that, in the early phase of COVID-19 pneumonia, routine screening could miss patients who were virus carriers. Highlighting travel history is of paramount importance for the early detection and isolation of severe acute respiratory syndrome coronavirus 2 cases.

Distribution and phylogenetic analysis of Dabieshan tick virus in ticks collected from Zhoushan, China.

Dabieshan tick virus (DBV) belongs to Phlebovirus and its pathogenicity to human and animals is unknown. To investigate the presence of Dabieshan tick virus in Zhoushan, 353 ticks were collected from May 2018 to October 2019. The detection result showed that the average prevalence rate among these samples was 30.3% (107 positives out of 353 samples), which means DBVs are widely distributed in tick populations in Zhoushan of China. In a phylogenetic analysis based on the nucleotide sequences of the L and S segments of the virus (ZS-DBS-2018 tick virus) in the study, it clustered with Dabieshan tick virus (KM817666.1, KM817733.1) with a 97.1% and 99.6% nucleotide identity, respectively. Further studies involving virus isolation are required to characterize Dabieshan tick virus and to expand the geographical distribution of the sampled ticks.

Distribution and genetic diversity of adeno-associated viruses in bats from coastal areas of Southeast China.

Bats are associated with several important zoonotic viruses from different families. One example includes adeno-associated viruses (AAVs), that are extensively detected in several animals, especially primates. To understand AAVs distribution and genetic diversity in the coastal areas of Southeast China, a total of 415 intestine samples were mostly collected from two provinces of southeast China, i.e., Zhejiang and Fujian province. Intestine samples from five bat species were collected for AAVs detection. The average prevalence rate for AAV detection among these samples was 18.6% (77 positives out of 415 samples) and ranged from 11.8 to 28.9% between the five bat species. This suggests that AAVs are widely distributed in diverse bat populations in southeast coastal areas of China. Based on the genome sequence of bat adeno-associated virus-CXC1(BtAAV-CXC1) from one AAV-positive sample, the genetic diversity of the detected AAVs were assessed and analyzed. Phylogenetic analysis revealed that BtAAV-CXC1 was comparatively distant to other major AAVs from mammals and non-mammals, with only a 52.9~64.7% nucleotide identity. However, they were phylogenetically closer to Rhinolophus sinicus bat adeno-associated virus (Rs-BtAAV1), with a 74.5% nt similarity. Partial analysis of the rep and cap overlapping open reading frame (ORF) sequences from bat AAV samples revealed 48 partial rep sequences and 23 partial cap sequences from positive samples shared 86.9 to 100% and 72.3 to 98.8% nucleotide identities among themselves, respectively. This suggests that the detected AAVs had a distinctly high genetic diversity. These findings led us to conclude that diverse AAVs may be widely distributed in bat populations from the southeast regions of China.

The Genome of the Great Gerbil Reveals Species-Specific Duplication of an MHCII Gene.

The great gerbil (Rhombomys opimus) is a social rodent living in permanent, complex burrow systems distributed throughout Central Asia, where it serves as the main host of several important vector-borne infectious pathogens including the well-known plague bacterium (Yersinia pestis). Here, we present a continuous annotated genome assembly of the great gerbil, covering over 96% of the estimated 2.47-Gb genome. Taking advantage of the recent genome assemblies of the sand rat (Psammomys obesus) and the Mongolian gerbil (Meriones unguiculatus), comparative immunogenomic analyses reveal shared gene losses within TLR gene families (i.e., TLR8, TLR10, and the entire TLR11-subfamily) for Gerbillinae, accompanied with signs of diversifying selection of TLR7 and TLR9. Most notably, we find a great gerbil-specific duplication of the MHCII DRB locus. In silico analyses suggest that the duplicated gene provides high peptide binding affinity for Yersiniae epitopes as well as Leishmania and Leptospira epitopes, putatively leading to increased capability to withstand infections by these pathogens. Our study demonstrates the power of whole-genome sequencing combined with comparative genomic analyses to gain deeper insight into the immunogenomic landscape of the great gerbil and its close relatives.

Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress.

We employed a stepwise selection model for investigating the dynamics of antibiotic-resistant variants in Escherichia coli K-12 treated with increasing concentrations of ciprofloxacin (CIP). Firstly, we used Sanger sequencing to screen the variations in the fluoquinolone target genes, then, employed Illumina NGS sequencing for amplicons targeted regions with variations. The results demonstrated that variations G81C in gyrA and K276N and K277L in parC are standing resistance variations (SRVs), while S83A and S83L in gyrA and G78C in parC were emerging resistance variations (ERVs). The variants containing SRVs and/or ERVs were selected successively based on their sensitivities to CIP. Variant strain 1, containing substitution G81C in gyrA, was immediately selected following ciprofloxacin exposure, with obvious increases in the parC SRV, and parC and gyrA ERV allele frequencies. Variant strain 2, containing the SRVs, then dominated the population following a 20× increase in ciprofloxacin concentration, with other associated allele frequencies also elevated. Variant strains 3 and 4, containing ERVs in gyrA and parC, respectively, were then selected at 40× and 160× antibiotic concentrations. Two variants, strains 5 and 6, generated in the selection procedure, were lost because of higher fitness costs or a lower level of resistance compared with variants 3 and 4. For the second induction, all variations/indels were already present as SRVs and selected out step by step at different passages. Whatever the first induction or second induction, our results confirmed the soft selective sweep hypothesis and provided critical information for guiding clinical treatment of pathogens containing SRVs.

Correction to: The polymorphism and geographical distribution of knockdown resistance of adult Anopheles sinensis populations in eastern China.

Please be advised that since publication of the original article [1] the authors have flagged that they omitted to provide the up-to-date version of Fig. 1 and, as such, the wrong version of Fig. 1 is present in the article.

The polymorphism and geographical distribution of knockdown resistance of adult Anopheles sinensis populations in eastern China.

BACKGROUND: Anopheles sinensis is one of the major malaria vectors in China and other southeast Asian countries, including Vietnam, Cambodia, Thailand. Vector control is considered to be the critical measure for malaria control, while the increasing prevalence of insecticide resistance caused by long-term use of insecticides, especially pyrethroids, is threatening the successful control of An. sinensis. In order to understand the underlying resistance mechanisms involved and molecular basis, the principal malaria vector, An. sinensis from Jiangsu and Anhui provinces, Southeast China, was investigated. METHODS: The adult Anopheles mosquitoes were sampled from multiple sites across Jiangsu and Anhui provinces, and sufficient mosquitoes collected from eleven sites for insecticide susceptibility bioassays. The DIIS4-DIIS6 region of the para-type sodium channel gene was amplified and sequenced, then multiple PCR and Taqman assays were used to assess the frequencies of kdr mutations at the target gene. RESULTS: In the present study, most of the adult An. sinensis populations were pyrethroids resistant, which indicated the presence of kdr resistance mutations in the para-type sodium channel gene. Sequence analyses demonstrated the kdr mutation existed at codon 1014 in Jiangsu and Anhui provinces. In adult An. sinensis, three mutant types (TTT L1014F, TTC L1014F, and TGT L1014C) of kdr alleles were detected, while no wild type (TTG L1014) was observed. The TTC L1014F mutation was first reported in Anhui province. CONCLUSIONS: The highly polymorphic kdr alleles were observed in all the adult An. sinensis populations, which suggested that in-depth studies are required for carrying on insecticide resistance monitoring and specific resistance mechanisms studying into establish effective long-term malaria vector control program in eastern China.

[Two-plasmid scarless genetic modification in Escherichia coli hfq and rne-710].

Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.

Characterization of a novel class A carbapenemase PAD-1 from Paramesorhizobium desertii A-3-ET, a strain highly resistant to ß-lactam antibiotics.

Although clinical antibiotic-resistant bacteria have attracted tremendous attention in the microbiology community, the resistant bacteria that persist in natural environments have been overlooked for a longtime. We previously proposed a new species Paramesorhizobium desertii, isolated from the soil of the Taklimakan Desert in China that is highly resistant to most ß-lactam antibiotics. To identify potential ß-lactamase(s) in this bacteria, we first confirmed the carbapenemase activity in the freeze-thawed supernatant of a P. desertii A-3-ET culture using the modified Hodge assay. We then identified a novel chromosome-encoded carbapenemase (PAD-1) in strain A-3-ET, using a shotgun proteomic analysis of the supernatant and genomic information. The bioinformatics analysis indicated that PAD-1 is a class A carbapenemase. Subsequent enzyme kinetic assays with purified PAD-1 confirmed its carbapenemase activity, which is similar to that of clinically significant class A carbapenemases, including BKC-1 and KPC-2. Because the location in which A-3-ET was isolated is not affected by human activity, PAD-1 is unlikely to be associated with the selection pressures exerted by modern antibiotics. This study confirmed the diversity of antibiotic-resistant determinants in the environmental resistome.

A1S_2811, a CheA/Y-like hybrid two-component regulator from Acinetobacter baumannii ATCC17978, is involved in surface motility and biofilm formation in this bacterium.

Two-component systems in Acinetobacter baumannii are associated with its virulence, drug resistance, motility, biofilm formation, and other characteristics. In this study, we used RecAb , a genetic engineering method, to investigate the function of A1S_2811 in A. baumannii strain ATCC17978. A1S_2811, a hypothetical hybrid sensor histidine kinase/response regulator, has four histidine-containing phosphotransfer domains, a CheA-like regulatory domain, and a CheY-like receiver domain at its C terminus. Compared with the ATCC17978 strain, both surface motility and biofilm formation at the gas-liquid interface decreased significantly in the A1S_2811 knock-out strain. The number of pilus-like structures and the amount of extrapolymeric substances on the cell surface also decreased in the A1S_2811 null strain. Transcription of abaI, which encodes an N-acylhomoserine lactone synthase in A. baumannii , decreased significantly in the A1S_2811 null strain, and supplementation with synthetic N-(3-oxodecanoyl) homoserine-l-lactone rescued the surface motility and biofilm formation phenotype in the null mutant. We speculate that A1S_2811 regulates surface motility and biofilm formation, not by regulating type IV pili-associated genes expression, but by regulating the chaperone/usher pili-associated csuA/ABCDE operon and the AbaI-dependent quorum-sensing pathway-associated A1S_0112-0119 operon instead.