Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It is noteworthy that tet(X4)-positive E. coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.

Seroprevalence of Toxoplasma gondii Infection in Patients of Intensive Care Unit in China: A Hospital Based Study.

The objective of this study was to estimate the seroprevalence of Toxoplasma gondii infection in 394 patients of intensive care unit (ICU) in a hospital between April 2010 and March 2012 and analyze the association between T. gondii infection and ICU patients according to the species of disease. Toxoplasma serology was evaluated by ELISA method using a commercially available kit. Data of patients were obtained from the patients, informants, and medical examination records. Seventy-four (18.78%) of 394 patients were positive for anti-T. gondii IgG antibodies demonstrating latent infection. Of these, the highest T. gondii seroprevalence was found in the age group of 31-45 years (27.45%), and the lowest was found in the age group of <30 years (12.5%). In addition, females (21.6%) had a higher seroprevalence than males (18.36%). With respect to the species of disease, the patients with kidney diseases (57.14%), lung diseases (27.84%), and brain diseases (24%) had high T. gondii seroprevalence. The present study represents the first survey of T. gondii seroprevalence in ICU patients in China, revealing an 18.78% seropositivity. Considering the particularities of ICU patients, molecular identification, genetic characterization, and diagnosis of T. gondii should be considered in future study.

Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics.

Outer membrane vesicles (OMVs) are well-characterized virulence factors produced by Gram-negative bacteria. Here, we isolated two clinical Acinetobacter baumannii strains, the multidrug-resistant A. baumannii (MDRAb) A38 and non-MDRAb 5806. Strain A38 produced more abundant OMVs than strain 5806 when cultured to the early stationary phase. The results from cell proliferation assays and real-time PCR analyses indicated that A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses compared with 5806 OMVs. Moreover, SDS-PAGE and LC-MS/MS analyses revealed that A38 OMVs contained more virulence factors, including Omp38, EpsA, Ptk, GroEL, hemagglutinin-like protein, and FilF. Taken together, the results of the present study suggest that MDRAb might produce abundant OMVs with more virulent factors facilitating the worse outcome, a finding that merits further study.

Differences in microglia activation between rats-derived cell and mice-derived cell after stimulating by soluble antigen of IV larva from Angiostrongylus cantonensis in vitro.

Angiostrongylus cantonensis is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats. Humans and mice are accidental hosts or named nonpermissive hosts. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningoencephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of the host to parasite during A. cantonensis invasion and development. Microglia is considered to be the key immune cell in the central nervous system like macrophage. To further understand the reasons for why mice and rats attain different outcomes in A. cantonensis infection, we set up the method to isolate and culture newborn rats' primary microglia and observe the activation of the microglia cells, comparing with mice microglia cell line N9. We treated cells with soluble antigen of the fourth larva of A. cantonensis (L4 larva) and measured mRNA levels of IL-1ß, IL-5, IL-6, IL-13, eotaxin, iNOS, and TNF-α by real-time PCR. The results showed that N9 expressed high mRNA level of IL-6, IL-1ß, TNF-α, iNOS, IL-5, IL-13, and eotaxin, but primary microglia only had IL-5, IL-13, and eotaxin mRNA level. It implies that microglia from rats and mice had different reaction to soluble antigen of A. cantonensis. Therefore, we supposed that microglia may play an immune modulation role during the brain inflammation induced by A. cantonensis.

Intranasal immunization with autolysin (LytA) in mice model induced protection against five prevalent Streptococcus pneumoniae serotypes in China.

In order to evaluate immunogenicity and protective efficacy of LytA from Streptococcus pneumoniae, we subcloned the full-length lytA-encoded autolysin (LytA) from 5 major pathogenic serotype isolates in China and obtained purified rLytA. Bioinformatics analysis showed that sequences of LytA were highly conserved in all strains we used in this work, and western blot analysis demonstrated that rLytAs from heterogeneous serotypes were cross-recognized by serum of mice infected with 23F strain SH137. Mice were intranasally immunized with purified rLytA, and serum anti-rLytA IgG, IgA and secretory IgA were elicited. More importantly, rLytA intranasal-immunized mice showed a significantly higher survival rate and lower bacterial carriage in response to infection by Streptococcus pneumoniae. The fact that mice immunized with rLytA from strain SH137 also had a higher survival rate after intraperitoneal injection of other four serotype strains of living S. pneumoniae suggested that it possessed cross-protection effect. Our study revealed that intranasal immunization with rLytA may protect mice against mucosal and systemic pneumococcal infection; hence, it was an attractive vaccine candidate.

The complete mitochondrial genomes of three cestode species of Taenia infecting animals and humans.

Mitochondrial (mt) genome sequences provide useful markers for investigating population genetic structures, systematics and phylogenetics of organisms. Although Taenia multiceps, T. hydatigena, and T. taeniaeformis are common taeniid tapeworms of ruminants, pigs, dogs, or cats, causing significant economic losses, no published study on their mt genomes is available. The complete mt genomes of T. multiceps, T. hydatigena, and T. taeniaeformis were amplified in two overlapping fragments and then sequenced. The sizes of the entire mt genome were 13700 bp for T. multiceps, 13489 bp for T. hydatigena, and 13647 bp for T. taeniaeformis. Each of the three genomes contains 36 genes, consisting of 12 genes for proteins, 2 genes for rRNA, and 22 genes for tRNA, which are the same as the mt genomes of all other cestode species studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T of the complete genomes are 71.3% for T. multiceps, 70.8% for T. hydatigena, and 73.0% for T. taeniaeformis. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. T. multiceps and T. hydatigena had two noncoding regions, but T. taeniaeformis had only one. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes revealed that T. multiceps, T. hydatigena, and T. taeniaeformis were more closely related to the other members of the Taenia genus, consistent with results of previous morphological and molecular studies. The present study determined the complete mt genome sequences for three Taenia species of animal and human health significance, providing useful markers for studying the systematics, population genetics, and molecular epidemiology of these cestode parasites of animals and humans.

Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis.

Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 degrees C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.

[A multicentre study on the pathogenic agents in 665 adult patients with community-acquired pneumonia in cities of China].

OBJECTIVE: To investigate the pathogenic causes of community-acquired pneumonia (CAP) in adult patients in China, the relation of previous antibiotic use and the Pneumonia Patient Outcome Research Team (PORT) classification to microbial etiology, and the prevalence of drug resistance of common CAP bacteria. METHODS: A prospective study was performed on 665 consecutive adult patients with CAP at 12 centers in 7 Chinese cities during one year. The etiology of pneumonia was considered if one of the following criteria was met: (1) valid sputum sample yielding one or more predominant strains; (2) blood cultures yielding a bacterial pathogen; (3) seroconversion, a > or = 4-fold increase or decrease titers of antibodies to Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila. Minimum inhibitory concentration (MIC) of respiratory tract isolates was determined using the agar dilution method. RESULTS: Pathogens were identified in 324/610 patients (53.1%) with valid serum samples and sputum cultures as follows: Mycoplasma pneumoniae (126, 20.7%), Streptococcus pneumoniae (63, 10.3%), Haemophilus influenzae (56, 9.2%), Chlamydia pneumoniae (40, 6.6%), Klebsiella pneumoniae (37, 6.1%), Legionella pneumophila (31, 5.1%), Staphylococcus aureus (23, 3.8%), Escherichia coli (10, 1.6%), Moraxella catarrhalis (8, 1.3%), Pseudomonas aeruginosa (6, 1.0%). Of 195 patients with a bacterial pathogen, an atypical pathogen was identified in 62 (10.2%) cases. The non-susceptibility rate of Streptococcus pneumoniae to penicillin, azithromycin, and moxifloxacin was 20.3%, 75.4% and 4.3% respectively. CONCLUSIONS: Atypical pathogens have important role in CAP, with Mycoplasma pneumoniae being the most common pathogen, and mixed infection of atypical pathogens with bacteria was found in 10.2% of the cases. Streptococcus pneumoniae and Haemophilus influenzae remain the most important bacteria for CAP. More than 75.0% of Streptococcus pneumoniae was resistant to macrolides and 20.3% was resistant to penicillin.

[A retrospective cohort study of the influence of time of hospital-acquired pneumonia onset on pathogen constitution].

OBJECTIVE: To study the influence of duration of hospitalization on etiologic agent and antibiotic-resistance of hospital-acquired pneumonia (HAP). METHODS: Cases of HAP were patients hospitalized in Fudan University Zhongshan Hospital, Ruijin Hospital, Beijing Hospital, Zhongshan University Affiliated Third Hospital, Guangzhou Medical College Affiliated Hospital and Guangdong People's Hospital. These patients were hospitalized from January 2001 to December 2003, and the diagnosis of HAP was made based on positive respiratory specimen cultures. Clinical data including time of HAP onset, severity of illness, risk factors, isolated bacteria and antimicrobial susceptibility were collected and analyzed. Statistical analysis was performed with the SPSS 12.0 software. RESULTS: A total of 562 cases of HAP were recruited, including 136 cases of early-onset pneumonia (time of onset < or = 5 d), 326 cases of middle-onset pneumonia (time of onset 6 - 14 d) and 100 cases of late-onset pneumonia (time of onset > or = 15 d). The rate of prior antibiotic use increased from 68.4% in the early-onset group to 88.0% in the late-onset group (P = 0.002); ICU admission increased from 29.4% to 46.0% (P = 0.03), and immunosuppression increased from 1.5% to 15% (P = 0.001). A total of 918 strains of bacteria were isolated, the most common pathogens being Pseudomonas aeruginosa (18.6%), Staphylococcus aureus (16.1%), Acinetobacter spp (16.1%), Klebsiella spp (14.4%) and Enterobacter spp (8.8%). Early-onset HAP were more commonly caused by Klebstella (18.3%), while the main etiologic agents for late-onset HAP were Pseudomonas aeruginosa (24.2%) and Methicillin-resistant Staphylococcus aureus (19.3%). The rates of pneumonia caused by Haemophilus and Streptococcus were 4.3% and 2.4% respectively in the early-onset cases, but none was found in late-onset cases. The antibacterial activity of ceftriaxone was influenced by duration of hospitalization, risk factors and severity of the disease. In less severe early-onset cases without risk factors, the sensitivity of ceftriaxone was 80%. But in severe late-onset cases, it was only 50%. CONCLUSIONS: There was significant difference in the pathogen constitution and antibiotic-resistance among early-onset, middle-onset and late-onset cases of HAP. The sensitivity of ceftriaxone was high in less severe early-onset cases without risk factors.

[Effect of specific immunoglobulin Y in the treatment of acute and chronic pharyngitis].

OBJECTIVE: To evaluate the effect and safety of the specific immunoglobulin Y (IgY) for treatment of acute and chronic pharyngitis. METHODS: Double-blind, randomized, placebo-controlled trial was conducted on 50 adults with acute pharyngitis. Experimental group received a 6 times-daily total 30 doses of IgY stomat-spray which contained specific immunoglobulin Y (titer = 512) prepared from the egg yolk of hens immunized with a variety of bacteria. Another open label trial included 50 patients, whose ages ranged from 21-69 years, including 25 cases of acute pharyngitis and 25 cases of chronic pharyngitis were also treated using IgY stomat-spray. The therapeutic effect were objectively evaluated 7 days later by the decreased scores based on both the symptoms and physical signs. If the symptom did not improve or became severe three days later, these patients with acute pharyngitis was inefficiency and antibiotic medicine would be added to them. RESULTS: In Double-blind trial, 8 cases (32%) received IgY had apparent effect with the decreased scores 5 or more than 5, 13 cases (52%) had effective with the decreased scores 3-4, and other 4 cases (16%) had inefficacy with the decreased scores only 2 or no more than 2. While in placebo-controlled group, only 2 (8%) cases had apparent effect, 5 (20%) cases showed effective and 18 (72%) cases had non-effect. The difference between the two groups was significant (chi 2 = 16.06, P < 0.01). In open label trial, 19 cases (38%) showed apparent effect, in which 14 cases were acute pharygitis. 23 cases (46%) had effective, in which 10 cases were acute pharyngitis. The left 8 cases (16%) had ineffective, in which one case was acute pharyngitis. There was significantly difference (chi 2 = 8.90, P < 0.05) between acute pharyngitis and chronic pharyngitis. An average of three months followup showed that there were no side effect or toxic effect and no allergic reaction. CONCLUSION: The IgY stomat-spray is a safe and effective agent in treating acute and chronic pharyngitis, especially for acute pharyngitis.

[An experimental study of the therapeutic effect of interleukin-2 and interleukin-12 with and without amphotericin B on pulmonary fungal infection].

OBJECTIVE: To study the therapeutic effect of interleukin-2 (IL-2) and interleukin-12 (IL-12) with and without amphotericin B on pulmonary fungal infection of mice. METHODS: A mouse model of pulmonary invasive aspergillus fumigatus (IPA) infection was established and the mice were divided into different groups, treated with IL-2 and IL-12 with and without amphotericin B. The survival number of mice in 15 days and the colony count of lung tissue in the different groups were observed. RESULTS: IL-2, IL-12 and amphotericin B showed synergistic effect in prolonging the survival of the infected mice and reducing the colony count in the lung tissue. CONCLUSION: IL-2 and IL-12 are effective adjuvant therapeutic agents in the immunosuppressed hosts.

[Study on the molecular mechanism of transferable multiple-antibiotic resistance in extended-spectrum beta-lactamase-producing clinical isolates].

OBJECTIVE: To investigate the molecular mechanism of transferable multiple-antibiotic resistance in extended-spectrum beta-lactamases (ESBLs) producing isolates. METHODS: Antibiotics susceptibility was tested by E-test method, and multi-resistance plasmids were screened and isolated by extracting transformant plasmids. Inserted gene Cassettes of class 1 integron were amplified and analyzed by polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Eight of the nine ESBL-producing plasmids were found to comprise class 1 integron sequence, of them 7 harbored 1 or 2 antibiotic resistant gene cassettes which encoding resistance to aminoglycosides (aacA4, aadA2 or aadA5), trimethoprim (dhfrA12 or dfrA17), rifampicin (arr-3) and chloramphenicol (cmlA6). The function of these gene cassettes corresponded to the resistance profiles of their electro-transformants. CONCLUSION: Multi-resistance gene cassettes located on plasmids and mediated by class 1 integron may play an important role in causing the development and dissemination of multiple-antibiotic resistance in ESBL-producing clinical isolates.