Characterization of antibiotic resistance genes and mobile elements in extended-spectrum ß-lactamase-producing Escherichia coli strains isolated from hospitalized patients in Guangdong, China.

AIM: This study aimed to investigate the high-resolution phenotypic and genotypic characterization of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli strains isolated from hospitalized patients to explore the resistance genes and mobile genetic elements (MGEs) involved in horizontal dissemination. METHODS: Between May and September 2021, a total of 216 ESBL-producing E. coli isolates were recovered from multiple departments. The identification of strains was performed using MALDI-TOF mass spectrometry and PCR, while antibiotic susceptibility testing was carried out using the Vitek 2 COMPACT system to determine resistance patterns, while PCR was used to detect different resistance genes and MGEs. In addition, a conjugation assay was performed to investigate the horizontal gene transfer of resistance genes. Selected isolates underwent whole-genome sequencing (WGS) using the Illumina MiSeq platform. RESULTS: A total of 216 out of 409 E. coli isolates recovered from a tertiary hospital were observed to be ESBL-producing, giving a carriage rate of 52.8%, as determined by phenotypic screening. The most frequent sources of ESBL-producing E. coli isolates were urine (129/216, 59.72%) and blood (50/216, 23.14%). The most prevalent ESBL genes identified were blaCTX-M (60.18%), blaTEM (40.27%), and blaSHV (18.05%). Three E. coli isolates were found to carry the genes blaNDM, mcr-1, and fosA3 genes. The most prevalent MGEs were IS26 (95.37%), Int (87.03%), and IncFIB (76.85%). WGS analysis of eight MDR E. coli strains revealed that these isolates belonged to eight different sequence types (STs) and serotypes and were found to harbor multiple plasmid replicons and virulence factors. CONCLUSION: This study highlights a high incidence of antibiotic resistance genes and MGEs associated with the dissemination of ESBLs and other resistance genes.

Photodynamic therapy treats acne by altering the composition of the skin microbiota.

BACKGROUND: Acne is the eighth-most prevalent inflammatory skin disease with no optimal treatment. Photodynamic therapy (PDT) is an effective treatment for severe acne. AIMS: The effect of PDT on the composition and diversity of skin microflora in severe acne patients was studied. MATERIALS AND METHODS: A total of 18 patients with severe acne and 8 healthy individuals were selected for this study. Patients were treated with 5-aminolevulinic acid-mediated PDT once a week three times in total; the skin microbiome was measured by 16S ribosomal RNA gene sequencing before and after treatment (1 week after each PDT). RESULTS: The microflora composition was different between healthy controls and patients, and between patients before and after treatment. Alpha diversity indices were lower in patients than those in control. There were 15 bacterial genera with high relative abundance that had noticeable changes during treatment. At the genus level,particularly Cutibacterium acnes (C. acnes formerly Propionibacterium acnes), there was no statistically significant difference among different group. The abundances of Staphylococcus epidermidis and Staphylococcus aureus were low. DISCUSSION: The microbial composition is different between severe acne patients acne patients and healthy individuals. The therapeutic efficacy of severe acne treated with PDT is associated with the composition and diversity of skin microbiota. CONCLUSION: The skin microbial composition changes after PDT treatment. PDT is an effective method for the treatment of severe acne.

Ecological mechanisms of sedimental microbial biodiversity shift and the role of antimicrobial resistance genes in modulating microbial turnover.

The mechanisms of phylogenetic turnover of microbial communities to environmental perturbations in sediments remain unclear. In this study, the molecular mechanisms of phylogenetic turnover, and impact of antibiotics and antibiotic resistance genes (ARGs) on the modification of microbial assemblages were unravelled. We investigated 306 ARGs, 8 transposases, and 4 integron integrases, bacteria, and eukaryotic diversity through high-throughput quantitative PCR and illumina sequencing, 21 antibiotics and 3 tetracycline byproducts. The freshwater and estuary ecosystems were mainly dominated by genus Sulfurovum and colonised by closely related species compared with the estuary (closeness centrality = 0.42 vs. 0.46), which was dominated by genus Mycobacterium. Eighty-six percent of the ecological process in the bacterial community was driven by stochastic processes, while the rest was driven by deterministic processes. Environmental-related concentrations of antibiotics (0.15-32.53 ng/g) stimulated the proliferation of ARGs which potentially modulated the microbial community assembly. ARG acquisition significantly (P < 0.001) increased eukaryotic diversity through protection mechanisms. ARGs showed complex interrelationships with the microbial communities, and phylum arthropods and Nematea demonstrated the strongest ARG acquisition potential. This study provides key insights for environmental policymakers into understanding the ecological impact of antibiotics and the role of ARGs in modulating the phylogenetic turnover of microbial communities and trophic transfer mechanisms.

A unique Levey-Jennings control chart used for internal quality control in human papillomavirus detection.

OBJECTIVE: The purpose of this study was to provide an updated estimate of the prevalences of different types of human papillomavirus (HPV) in females in Chaoshan District and to establish an internal quality control (IQC) method for excluding false-positive results in HPV detection by using the Levey-Jennings control chart. METHOD: HPV types were detected in 23,762 cervical samples by using PCR membrane hybridization. The means and standard deviations (SDs) of the positive rates were calculated, the Levey-Jennings chart was plotted, and the rules for "out of control" and "warning" were established. A set of standardized IQC for HPV DNA tests was developed based on the values and Levey-Jennings charts. RESULT: In 466 batches, the positive rate exceeded the 1 + 2SD rule 24 times, but there was no consecutive exceedance, which was considered "in control". When the positive rate exceeded the 1 + 3SD rule 8 times with consecutive exceedance, it was considered "out of control". Further examination revealed that detections showing "out of control" had an undesirable random error, indicating that contamination may occur due to improper operation. CONCLUSION: This unique Levey-Jennings control chart is a practical method for eliminating false-positive results in HPV DNA detection and should be widely applicable in molecular diagnostic laboratories.

Surveillance of SARS-CoV-2 antibodies of patients in the local affected area during Wuhan lockdown.

BACKGROUND: Serosurveillance is crucial in estimating the range of SARS-CoV-2 infections, predicting the possibility of another wave, and deciding on a vaccination strategy. To understand the herd immunity after the COVID-19 pandemic, the seroprevalence was measured in 3062 individuals with or without COVID-19 from the clinic. METHODS: The levels of SARS-CoV-2 antibody IgM and IgG were measured by the immuno-colloidal gold method. A fusion fragment of nucleocapsid and spike protein was detected by a qualitative test kit with sensitivity (89%) and specificity (98%). RESULTS: The seroprevalence rate for IgM and IgG in all outpatients was 2.81% and 7.51%, respectively. The sex-related prevalence rate of IgG was significantly higher (P < 0.05) in women than men. The highest positive rate of IgM was observed in individuals < 20 years of age (3.57%), while the highest seroprevalence for IgG was observed in persons > 60 years of age (8.61%). Positive rates of IgM and IgG in the convalescent patients were 31.82% and 77.27%, respectively, which was significantly higher than individuals with suspected syndromes or individuals without any clinical signs (P < 0.01). Seroprevalence for IgG in medical staff was markedly higher than those in residents. No significant difference of seroprevalence was found among patients with different comorbidities (P > 0.05). CONCLUSIONS: The low positive rate of the SARS-CoV-2 IgM and nucleic acid (NA) test indicated that the SARS-CoV-2 outbreak is subsiding after 3 months, and the possibility of reintroduction of the virus from an unidentified natural reservoir is low. Seroprevalence provides information for humoral immunity and vaccine in the future.

Incidence and molecular characterization of ESBL-producing and colistin-resistant Escherichia coli isolates recovered from healthy food-producing animals in Pakistan.

OBJECTIVES: To investigate the occurrence and molecular features of ESBL-producing and colistin-resistant Escherichia coli isolates recovered from healthy food-producing animals in Pakistan. METHODS: A total of 153 E. coli isolates were recovered from 250 faecal samples collected from livestock and poultry. The antibiotic susceptibility, resistant determinants and mobile genetic elements were determined for all the isolates. The clonal relatedness was analysed by MLST. Plasmids harbouring, localization and transferability of mcr-1 gene were carried out by Southern hybridization, S1-PFGE and transconjugation. RESULTS: Out of 153 E. coli strains, 49.01% isolates were ESBLs producers, whereas 18.95% were resistant to colistin and 84.31% of the isolates. Multidrug resistance was found in 84% of the isolates. The ESBL-producing E. coli in buffaloes, cattle, sheep, goat and broilers faecal samples were 60%, 74%, 54%, 50% and 68%, respectively. Among the ESBLs genes, blaCTX-M was the most prevalent group detected in 98.66%, while only mcr-1 of the colistin-resistant genes could be PCR amplified in 29 isolates. The common MGEs found were ISECP1 (35.13%), ISCR1 (33.78%), ISApl1 (20.27%) and Inti1 (58.10%). The most predominant Inc. types found were IncFIB 46.66%, followed by IncFIA 30.66%, IncFIC 26.66%, IncFrepB 26.66%, IncHI2 26.66%, IncP 22.66% and IncX4 21.33%. The most frequent sequence type detected was ST58. Southern blot and S1-PFGE confirmed the plasmid harbouring of mcr-1 gene. CONCLUSION: The co-occurrence of mcr-1 and ESBLs-encoding genes, along with MGEs in E. coli from healthy food animals in Pakistan, is a major concern. SIGNIFICANCE AND IMPACT OF STUDY: Antimicrobial resistance can be transferred from animals to humans by direct contact or via the food chain and environment. The prevalence and co-occurrence of ESBL and colistin resistance genes from food-producing animals is rare in Pakistan. To our knowledge, this is the first report to find ESBLs and mcr-1-harbouring E. coli from the faecal samples of the healthy food-producing animals in Pakistan. The presence of ARGs in association with MGEs, co-harbouring the virulence factors, as determined in the current study, is a severe threat to livestock and the human community as it has horizontally and food web transferability.

Aberrant cytokine expression in COVID-19 patients: Associations between cytokines and disease severity.

Cytokines play pleiotropic, antagonistic, and collaborative in viral disease. The high morbidity and mortality of coronavirus disease 2019 (COVID-19) make it a significant threat to global public health. Elucidating its pathogenesis is essential to finding effective therapy. A retrospective study was conducted on 71 patients hospitalized with COVID-19. Data on cytokines, T lymphocytes, and other clinical and laboratory characteristics were collected from patients with variable disease severity. The effects of cytokines on the overall survival (OS) and event-free survival (EFS) of patients were analyzed. The critically severe and severe patients had higher infection indexes and significant multiple organ function abnormalities than the mild patients (P < 0.05). IL-6 and IL-10 were significantly higher in the critically severe patients than in the severe and mild patients (P < 0.05). IL-6 and IL-10 were closely associated with white blood cells, neutrophils, T lymphocyte subsets, D-D dimer, blood urea nitrogen, complement C1q, procalcitonin C-reactive protein. Moreover, the IL-6 and IL-10 levels were closely correlated to dyspnea and dizziness (P < 0.05). The patients with higher IL-10 levels had shorter OS than the group with lower levels (P < 0.05). The older patients with higher levels of single IL-6 or IL-10 tended to have shorter EFS (P < 0.05), while the patients who had more elevated IL-6 and IL-10 had shorter OS (P < 0.05). The Cox proportional hazard model revealed that IL-6 was the independent factor affecting EFS. IL-6 and IL-10 play crucial roles in COVID-19 prognosis.

Establishing and evaluating an auto-verification system of thalassemia gene detection results.

The manual verification of gene tests is time-consuming and error prone. In this study, we try to explore a high-efficiency, clinically useful auto-verification system for gene detection of thalassemia. A series of verification elements were rooted in the auto-verification system. Consistency check was applied initially as one of the essential elements in our study. One hundred twenty-four archived cases were used to choose the consistency-check rules' indices from routine blood examination and hemoglobin electrophoresis by the receiver operating characteristic curves. Rule 1 and rule 2 established by the chosen indices were compared by their passing rate, consistency with manual validation, and error rate. Finally, 748 cases were used for verifying the system's feasibility by evaluating the passing rate, turn-around time (TAT), and error rate. The rule 2 had a higher passing rate (67.7% vs. 50.8%) and consistency (0.623 vs. 0.364) than the rule 1 with an error rate of zero. In a "live" valuation, the auto-verification system can reduce the TAT and error rate of verification by 51.5% and 0.13%, respectively, with a high passing rate of 82.8%. The auto-verification system for gene detection of thalassemia in this study can shorten the validation time, reduce errors, and enhance efficiency.

Th17 cytokine profiling of colorectal cancer patients with or without enterovirus 71 antigen expression.

OBJECT: Th17 cytokines have been identified in several types of human cancers. In this pilot study, the expression of Th17 cytokines profiling in enteroviruses 71 (EV71) associated colorectal cancer (CRC) were explored. METHODS: 66 patients with CRC were enrolled in this study; immune- histochemical analyses were performed on cancerous tissues and adjacent non- cancerous tissues of the patients. Serum Th17 cytokines of CRC patients and healthy controls were measured using a Luminex 200 analyzer. RESULTS: Cancerous tissues had more positive EV71 antigen expression than adjacent non- cancerous tissues. In TNM II-III CRC, 59.9% of cancerous tissues were observed to be EV71 positive; on the contrary, 65.2% of the adjacent non- cancerous epithelium was EV71 negative. In TNM I CRC, all adjacent non- cancerous epithelium was virus negative, but in TNM IV, half of adjacent non- cancerous tissues were virus positive. Serum IL-10 were significantly higher in CRC patients than in healthy controls, and IL-10 concentrations in the EV71 positive group were higher than those of the EV71 negative group, with the highest IL-10 levels being observed in CRC patients with strong positive group (P < 0.05). Similar results were found for IL-21 and IL-23. IL-17 levels were higher in CRC patients than in healthy controls, there was no significant difference in IL-17 between the viral positive and viral negative groups (P > 0.05). CONCLUSION: Persistent existing EV71 viral antigens in intestinal tissues are positively associated with TNM III/IV CRC. EV71 latent infection recruits Th17 cells in the colorectal tumor site, stimulating Th17 cytokine production that closely associated with CRC carcinogenesis.

Serum and exosomal miR-122 and miR-199a as a biomarker to predict therapeutic efficacy of hepatitis C patients.

MicroRNA (miRNA), which has been shown to correlate with liver functions, has been proposed as a new biomarker reflecting liver injury. The aim of the study was to investigate miRNA-122 (miR-122) and mir-RNA-199a (miR-199a) as a biomarker for predicting therapeutic efficacy in hepatitis C (HepC) patients. A total of 47 HepC 1b patients and 16 healthy subjects were enrolled in the study. Serum and exosomal mir-RNAs and other conventional biomarkers reflecting liver function were evaluated. The miR-122 levels in serum (miR-122ser ) and exosomes (miR-122exo ) were significantly lower in the Hepatitis C virus (HCV) genotype 1b patients than in the normal controls, but these levels were higher compared to the non-genotype 1b group. The mean miR-122ser level in the sustained virological response (SVR) group was significantly higher than that in the non-response (NR) group (P < 0.01), and the miR-122exo level in the SVR group was also higher than that in the NR group (P > 0.05), although this difference was not significant. miR-199a levels showed similar trends with the miR-122 levels in serum and exosomes. HCV RNAser was negatively correlated with the miR-122ser (r = -0.473, P = 0.004) and miR-122exo (r = -0.424, P = 0.009) levels. miR-122ser levels were positively associated with miR-199aser levels (r = 0.453, P = 0.002). Univariate and multivariate regression analyses reveal that the miR-122ser levels and ALT/AST ratio demonstrated a predictive value in evaluating patient outcomes. Serum miR-122 and miR-199a are potential biomarkers that reflect therapeutic efficacy.

Circulating microRNAs as a biomarker to predict therapy efficacy in hepatitis C patients with different genotypes.

BACKGROUND: Hepatitis C virus (HCV) genotype exerts a major influence on therapeutic response; however, the underlying mechanisms remain unclear. The aim of the study is to investigate the circulating microRNAs as the biomarkers to predict the response to therapy in chronic hepatitisC patients (HepC) with different genotypes. METHODS: HepC patients were separated into 4 groups by genotype, healthy individuals were enrolled as the control. microRNA-122 (miR-122), microRNA-155 (miR-155) and HCV RNA in serum and exosome were measured, associations between microRNAs, viral load and other conventional biomarkers were analyzed. RESULTS: Serum and exosomal HCV RNA in genotype 6a group was highest, followed by genotype 3a/2a, and in genotype 1b were the lowest. The significant correlations existed between exosomal HCV RNA and serum HCVRNA. MiR-122, both in serum (miR-122ser) and in exosome (miR-122exo), was higher in normal control than in HCV group. Specifically, miR-122exo were significantly higher in genotype 1b than other genotype groups (p < 0.05). On the contrary, miR-155exowas significantly lower in genotype 1b than in other groups (p < 0.05 for both). A strongly positive association was found between miR-122/155 and HCV viral load in patients with various genotypes. Higher miR-122ser at the start of therapy predicts a better outcome. CONCLUSIONS: Expression of miR-122/155 differ in each genotypes, miR-122ser could be independent factor affecting the therapy efficacy, which had higher diagnostic value in predicting HCV outcome.

Circulating tight junction proteins mirror blood-brain barrier integrity in leukaemia central nervous system metastasis.

The aim of this study was to evaluate the clinical significance of circulating tight junction (TJ) proteins as biomarkers reflecting of leukaemia central nervous system (CNS) metastasis. TJs [claudin5 (CLDN5), occludin (OCLN) and ZO-1] concentrations were measured in serum and cerebrospinal fluid (CSF) samples obtained from 45 leukaemia patients. Serum ZO-1 was significantly higher (p < 0.05), but CSF ZO-1 levels were not significantly higher in the CNS leukaemia (CNSL) compared to the non-CNSL. The CNSL patients also had a lower CLDN5/ZO1 ratio in both serum and CSF than in non-CNSL patients (p < 0.05). The TJ index was negatively associated with WBCCSF , ALBCSF and BBB values in leukaemia patients. Among all of the parameters studied, CLDN5CSF had the highest specificity in discriminating between CNSL and non-CNSL patients. Therefore, analysing serum and CSF levels of CLDN5, OCLN and the CLDN5/ZO1 ratio is valuable in evaluating the potential of leukaemia CNS metastasis. Copyright © 2016 John Wiley & Sons, Ltd.

Expiration Date Prediction of Biocontrol Agent Prepared with Bacillus subtilis B579 Using the Accelerated Aging Method.

The expiration date of biopesticidal products is an essential feature of their use and storage. In the present work, the expiration date of biocontrol agent was predicted using the accelerated aging method. The available bacteria in Bacillus subtilis B579 biocontrol agent were 3.7 ± 0.2 × 1011 CFU/g. It is calculated that the expiration date of the agent was about 17 months at 25°C. During this period, the available bacteria retained more than 90% of the value in the initial product. Thus, this work suggests the expiration date of biocontrol agents composed with spores could be estimated using the accelerated aging method.

The use of human umbilical cord blood serum is beneficial for the continuous production of hepatitis C virus.

In this study we investigated if human umbilical cord blood serum (CBS) is a suitable replacement for foetal bovine serum (FBS) in cultures of human hepatoma cell line Huh7.5, particularly regarding its capacity to maintain high growth rates, differentiation status and its ability to support robust hepatitis C virus (HCV) infection. Generally, CBS-cultured Huh7.5 cells remained comparable to FBS-cultured cells, and proliferated equally well. Albumin secretion, a hepatocyte differentiation marker, had increased 8x in CBS; however, most other hepatocyte markers we tested had not changed. Surprisingly, CBS-cultured cells were able to sustain very high levels of HCV production, and HCV infection in CBS-cultured cells did not induce cell lysis, which is typically seen in HCV-infected cells cultured in FBS. We discuss some of the differences between CBS, adult human serum and FBS that may explain the differences observed.

Viral etiology, clinical and laboratory features of adult hemophagocytic lymphohistiocytosis.

Secondary hemophagocytic lymphohistiocytosis (SHLH) is a potentially fatal hyperinflammatory syndrome with a heterogeneous etiology and has nonspecific clinical and laboratory findings. The diagnosis and treatment of adult SHLH is challenging because the etiology of the disease is difficult to identify, and the majority of reported cases are pediatric patients. The aim of this study was to describe the etiology, clinical characteristics, and outcomes of adult SHLH. Fifty-four adult patients who fulfilled the criteria of SHLH were enrolled in the study. Viral etiology, blood biomarkers, and clinical manifestations of SHLH were analyzed in these patients. Twenty-four SHLH patients had viraemia, whereas 30 SHLH patients were secondary to other diseases. Epstein-Barr virus (EBV) was the most common virus that associated SHLH among all viruses studied. Severe SHLH patients with EBV-viraemia presented significantly high levels of ferritin, lactate dehydrogenase, aspartate transaminase (AST), and alanine transaminase (ALT). Positively relationships existed between EBV DNA titers and levels of AST and ALT (P < 0.05). The prognosis of SHLH patients with EBV viraemia was worse than that of non-EBV SHLH and non-viral SHLH. Our data reveal that EBV is the major pathogen in virus-associated SHLH, and EBV load influence disease development in SHLH patients with EBV infection that prognosis is worse than other viruses associated SHLH.

Risk factors for the evaluation of potential central nervous system metastasis in Burkitt's lymphoma: a case study and literature review.

Burkitt's lymphoma (BL) is a malignancy of B lymphocytes. The rapid growth rate and frequent systemic spread result in most patients presenting with advanced disease at diagnosis. Cerebrospinal fluid cytology is the gold standard (with very high accuracy) for diagnosing BL central nervous system (CNS) metastasis; however, the low sensitivity of this method limits its clinical applications. Here, we report a case of BL with CNS metastasis. The levels of vascular endothelial growth factor (VEGF)-A and VEGF-C in the serum and cerebrospinal fluid were used to evaluate the status of BL remission and recurrence. Comparisons were made between VEGF and the other risk factors used in evaluating CNS metastasis. Although not in strict accordance, VEGF levels mirrored the disease course. Therefore, VEGF may reflect the status of BL CNS metastasis. Understanding the role of VEGF in CNS metastasis may help to improve the staging and risk classification of BL as well as the investigation of targeted therapy.

The prognostic significance of serum and cerebrospinal fluid MMP-9, CCL2 and sVCAM-1 in leukemia CNS metastasis.

Metastasis to the central nervous system (CNS) is the primary obstacle in leukemia treatment. Matrix metalloproteinase-9 (MMP-9), chemokine ligand-2 (CCL2) and soluble vascular adhesion molecule-1 (sVCAM-1) play crucial roles in tumor cell adhesion, motivation and survival, but their roles in leukemia CNS metastasis remain to be elucidated. We investigated the prognostic significance of serum and cerebrospinal fluid (CSF) MMP-9, CCL2 and sVCAM-1 in leukemia patients to explore their potential as predictive biomarkers of the development of CNS leukemia (CNSL). MMP-9, CCL2 and sVCAM-1 were measured in paired CSF and serum samples collecting from 33 leukemia patients with or without CNS metastasis. Other risk factors related to CNSL prognosis were also analyzed. sVCAM-1Serum and CCL2Serum/CSF were significantly higher in the CNSL group than in the non-CNSL group and the controls (p < 0.05). MMP-9Serum was insignificantly lower in the CNSL group than in the non-CNSL group and the controls (p > 0.05). No differences were found for the sVCAM-1Serum, CCL2Serum, and MMP-9Serum levels between non-CNSL patients and controls (p > 0.05). MMP-9CSF was significantly higher in the CNSL group than both the non-CNSL and the control groups (p < 0.05). The indexes of sVCAM-1, CCL2, and MMP-9 in the CNSL group were lower than in the controls (p < 0.05). Positive correlations were determined between the MMP-9CSF and the ALBCSF/BBB value/WBCCSF, between sVCAM-1Serum and the WBCCSF/BBB value. Negative correlations existed between MMP-9Serum and the ALBCSF/BBB value/WBCCSF, and between the CCL2 index and ALBCSF. sVCAM-1Serum was positively associated with event-free survival (EFS), and patients with higher levels of ALBCSF, MMP-9CSF/Serum, CCL2CSF/Serum, and sVCAM-1CSF/Serum had shorter EFS. MMP-9CSF, CCL2CSF and sVCAM-1CSF are the first three principal components analyzed by cluster and principal component analysis. Our data suggest that MMP-9, CCL2 and sVCAM-1 in the CSF may be more potent than serum in predicting the possibility of leukemia metastatic CNS and the outcome of CNSL patients.

A recombinant begomovirus resulting from exchange of the C4 gene.

A begomovirus isolated from Malvastrum coromandelianum and tomato originating from Yunnan province (China) was shown to be representative of a new begomovirus species, for which the name tomato leaf curl Yunnan virus (TLCYnV) is proposed. TLCYnV has high levels of sequence identity to tomato yellow leaf curl China virus (TYLCCNV) across the whole genome, except for sequences encompassing the C4 gene. Agrobacterium-mediated inoculation showed TLCYnV to be highly infectious to a range of plant species but poorly infectious to M. coromandelianum. In contrast to TYLCCNV, TLCYnV was shown to infect tomato in the absence of a betasatellite. In field-collected samples, TLCYnV was identified most frequently in tomato in which it was not associated with a betasatellite. Transgenic expression in Nicotiana benthamiana showed that the C4 protein of TYLCCNV did not induce developmental abnormalities, whereas the C4 of TLCYnV induced severe developmental abnormalities, reminiscent of virus symptoms. The genome of TLCYnV was shown to be significantly less methylated in plants than that of TYLCCNV and the C4 protein of TLCYnV was shown to suppress post-transcriptional gene silencing and transcriptional gene silencing more effectively than the C4 of TYLCCNV. The results indicate that TLCYnV evolved from TYLCCNV by recombination, acquiring a more virulent C4, allowing it to dispense with the requirement for a betasatellite. The implications of these findings in relation to the evolution of monopartite begomoviruses are discussed.

Antibiotic resistance in Pseudomonas aeruginosa is associated with decreased fitness.

The number of clinical specimens containing ß-lactam-resistant Pseudomonas aeruginosa isolates is increasing. However, whether resistance is associated with reduced fitness is still uncertain in clinical Pseudomonas aeruginosa isolates. In this study, we aimed to determine whether ß-lactam resistance conferred a fitness cost in Pseudomonas aeruginosa. Growth rate, extracellular slime production, elastase activity, proteolytic activity, LasA protease activity, biofilm formation, and pairwise in vitro competition experiments were investigated in a collection of 11 isogenic, ß-lactam-susceptible and -resistant (≥8-fold increase in minimum inhibitory concentration (MIC)) pairs of P. aeruginosa clinical isolates; each pair was recovered from a different patient treated with ß-lactam antibiotics. All ß-lactam-resistant Pseudomonas aeruginosa isolates showed a significant reduction in elastase activity. In addition, 90.9% (10/11) of ß-lactam-resistant Pseudomonas aeruginosa isolates were associated with markedly lower growth rate and proteolytic activity, and 81.8% (9/11) of ß-lactam-resistant Pseudomonas aeruginosa isolates had less extracellular slime production, compared to susceptible isolates. Meanwhile, LasA protease activity and biofilm formation ability were variable among isolates. Pairwise in vitro competition experiments showed that 72.7% (8/11) of ß- lactam-susceptible strains could outgrow resistant strains. In conclusion, resistance development with ß-lactam exposure confers a fitness cost, resulting in a decreased invasion potential, while the effect on viability varied. Thus, the potential for the dissemination of ß-lactam-resistant Pseudomonas aeruginosa clinical isolates should not be underestimated.

Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus in the family Geminiviridae, which causes severe losses in tomato production in tropic and subtropic regions. METHODS: The purified TYLCV virions were used as the immunogen to produce monoclonal antibodies (MAbs) using the hybridoma technology. MAb-based dot enzyme-linked immunosorbent assay (dot-ELISA) and direct tissue blot immunoassay (DTBIA) were developed for sensitive, simple, and rapid detection of TYLCV in field tomato and whitefly (Bemisia tabaci) samples collected from TYLCV prevalent provinces in China. RESULTS: Using the hybridoma technology, six murine MAbs (1C4, 8D10, 6E3, 2F2, 3F4 and 4G3) against TYLCV were prepared. Using the MAb 1C4, dot-ELISA and DTBIA were then established for detecting TYLCV in field tomato and whitefly samples collected from TYLCV prevalent provinces in China. The dot-ELISA could detect TYLCV in infected tissue crude extract diluted at 1:5,120 (w/v, g mL-1), and in viruliferous whitefly homogenate diluted at 1:128 (individual whitefly/µL), respectively. Field tomato samples (n=487) and whitefly samples (n=110) from TYLCV prevalent districts in China were screened for the presence of TYLCV using the two developed methods, and the results were further confirmed by PCR and nucleotide sequencing. The survey revealed that TYLCV is widespread on tomato plants in Zhejiang, Shandong and Henan provinces in China. CONCLUSIONS: The developed dot-ELISA is very suitable for the routine detection of TYLCV in field tomato and whitefly samples, and the DTBIA is more suitable for the routine detection of TYLCV in large-scale tomato plant samples collected from TYLCV prevalent areas.