Shiwei Liu

Coexistence of blaNDM-1 with the Prevalent blaOXA23 and blaIMP in Pan-drug Resistant Acinetobacter baumannii Isolates in China

To the Editor—The emergence of New Delhi metallo-beta-lactamase–1 (NDM1) in India, Pakistan, and the UK has sparked great fear of the threat posed by resistant microbial strains and by the use of antibiotics worldwide [1]. Thus far, NDM-1 carbapenemase has been detected in several countries in a diverse group of bacteria. Acinetobacter baumannii has emerged as one of the most troublesome pathogens for health care institutions globally. Its clinical significance has been propelled by its remarkable capability to upregulate or acquire resistance determinants, making it one of the most important organisms threatening the current antibiotic era. In Chinese hospitals, A. baumannii is the most common genus of carbapenemresistant bacteria [2]. We screened carbapenem-resistant A. baumannii for the presence of blaNDM-1 and successfully detected a blaNDM-1–positive strain, to our knowledge the first such strain of A. baumannii isolated in China. In addition to carbapenem, this isolate is resistant to various types of antibiotics—consistent with what has been observed in other countries [1]. Resistance to carbapenem is mainly mediated by 4 classes of carbapenemases among different bacterial species [3]. The carbapenemases found in A. baumannii thus far belong to either the OXA class D family of serine b-lactamases or to the IMP/VIM class B family of metallo-b-lactamases [4]. Polymerase chain reaction screening was performed to identify OXA type (OXA-23, OXA-24, and OXA-58–like) and the IMP/VIM genes. Of the 122 A. baumannii isolates from Chinese patients, 115 (94.2%) were positive for OXA-23 and 66 (54.0%) were positive for IMP. No other carbapenemase genes were detected. Sequencing of blaOXA-23 and blaIMP genes showed 100% identity with previously reported genes. All the IMP-positive isolates were also positive for OXA-23. The isolates positive for both OXA-23 and IMP showed increased resistance to most antibiotics (Table 1). Interestingly, the NDM1–positive isolate was also positive for both OXA-23 and IMP. The blaNDM-1– positive strain was more resistant to antibiotics than were strains that harbor both OXA-23 and IMP. However, we also found that the NDM-1–positive strain was susceptible to several fluoroquinolone antibiotics and to polymyxin B. Hospital-acquired infections with A. baumannii have been reported worldwide [5]. The success of the organism has been attributed to its ability for long-term survival in a hospital environment and its ability to rapidly acquire resistance to antibiotics. Although the exact mechanisms remain to be further defined, the emergence of blaNDM-1 in A. baumannii, and its coexistence with other carbapenemase genes, will seriously limit future therapeutic options.

High Severity and Fatality of Human Infections With Avian Influenza A(H7N9) Infection in China

Severe infection by a novel influenza virus, distinct from the circulating human influenza A virus, in humans usually heralds a sporadic pattern of severehuman infection or an influenza pandemic [1]. Accordingly, the discovery of the novel avian influenza A(H7N9) virus is of great public health significance [2]. Because this virus has not been detected previously in humans or in animals, many urgent questions and global public health concerns need to be addressed [3]. Study of the early cases could provide key

Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.

Confirming Ebola virus disease (EVD), a deadly infectious disease, requires real-time RT-PCR, which takes up to a few hours to yield results. Therefore, a rapid diagnostic assay is imperative for EVD diagnosis. A rapid nucleic acid test based on recombinase polymerase amplification (EBOV-RPA) was developed to specifically detect the 2014 outbreak strains. The EBOV-RPA assay was evaluated by testing samples from suspected EVD patients in parallel with RT-PCR. An EBOV-RPA, which could be completed in 20 min, was successfully developed. Of 271 patients who tested positive for Ebola virus by RT-PCR, 264 (sensitivity: 97%, 95% CI: 95.5–99.3%) were positive by EBOV-RPA; 101 of 104 patients (specificity: 97%, 95% CI: 93.9–100%) who tested negative by RT-PCR were also negative by EBOV-RPA. The sensitivity values for samples with a Ct value of <34, which accounted for 95.59% of the samples, was 100%. Discordant samples positive by RT-PCR but negative by EBOV-RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%, consistent with those of RT-PCR. The EBOV-RPA assay showed 97% sensitivity and 97% specificity for all EVD samples tested, making it a rapid and sensitive test for EVD diagnosis.

Large-scale identification of small noncoding RNA with strand-specific deep sequencing and characterization of a novel virulence-related sRNA in Brucella melitensis

Brucella is the causative agent of brucellosis, a worldwide epidemic zoonosis. Small noncoding RNAs (sRNAs) are important modulators of gene expression and involved in pathogenesis and stress adaptation of Brucella. In this study, using a strand-specific RNA deep-sequencing approach, we identified a global set of sRNAs expressed by B. melitensis 16M. In total, 1321 sRNAs were identified, ranging from 100 to 600 nucleotides. These sRNAs differ in their expression levels and strand and chromosomal distributions. The role of BSR0441, one of these sRNAs, in the virulence of B. melitensis 16M was further characterized. BSR0441 was highly induced during the infection of macrophages and mice. The deletion mutant of BSR0441 showed significantly reduced spleen colonization in the middle and late phases of infection. The expression of the BSR0441 target mRNA genes was also altered in the BSR0441 mutant strain during macrophage and mice infection, which is consistent with its reduced intracellular survival capacity. In summary, Brucella encodes a large number of sRNAs, which may be involved in the stress adaptation and virulence of Brucella. Further investigation of these regulators will extend our understanding of the Brucella pathogenesis mechanism and the interactions between Brucella and its hosts.

Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples

A rapid and sensitive recombinase polymerase amplification (RPA) assay, Bruce-RPA, was developed for detection of Brucella. The assay could detect as few as 3 copies of Brucella per reaction within 20 min. Bruce-RPA represents a candidate point-of-care diagnosis assay for human brucellosis.

Genome Sequence of an OXA23-Producing, Carbapenem-Resistant Acinetobacter baumannii Strain of Sequence Type ST75

The increase of Acinetobacter baumannii resistance to carbapenems is of great concern. OXA23 is one of the most prevalent carbapenemases of A. baumannii that causes outbreaks. Here, we announce the genome sequence of an OXA23-producing A. baumannii strain assigned ST75, a newly emerged sequence type harboring carbapenemase.

Traditional Chinese Medicine: An Alternative Treatment Option for Refractory Recurrent Urinary Tract Infections

TO THE EDITOR—With an annual incidence of approximately 150 million cases worldwide, urinary tract infection (UTI) represents one of the most common bacterial infections. Approximately 40% of women experience at least 1 symptomatic UTI during their lifetime, with approximately 25% suffering recurrent UTIs (RUTIs) [1]. Repeated antibiotic courses are often prescribed for treating and preventing RUTIs, resulting in multidrug and pandrug-resistant pathogens [2]. Uropathogenic Escherichia coli (UPEC) is responsible for 75%–90% of uncomplicated UTIs. Because of the multidrug and pandrug resistance of UPEC, these RUTIs can become refractory to antibiotic treatment. Effective alternative and complementary treatments will be valuable for these RUTIs. Traditional Chinese medicine (TCM), also termedherbalmedicine, has been extensively used in China for thousands of years to treat diverse pathologies, including chronic and refractory infectious diseases [3]. The feasibility of TCM to treat refractory RUTI has not been well defined. We evaluated the effectiveness of TCM for RUTI caused by pandrug-resistant bacterial pathogens. Thirty-four female patients (average age, 51.6 ± 15.4 years) with uncomplicated RUTI (defined as ≥3 microbiologically documented episodes of symptomatic UTI during the last year or 2 episodes during the last 6 months) were recruited. The annual incidence average was 6.6 ± 2.5 UTIs. UPEC strains were isolated from patients’ urine and were resistant to at least 8 antibiotics (pandrug resistance). They were treated with antibiotics at Western medicine hospitals but proved to be refractory to the treatment. These patients were treated with a 4week course of TCM consisting of 10 herbs (Rhizoma Anemarrhenae, Cortex Phellodendri Chinensis, Angelica sinensis, Rehmannia glutinosa Libosch, Wolfiporiacocos, Salvia miltiorrhiza, Rhubarb, Polygonum aviculare L., Dianthus superbus, and Talcum). After 2 weeks, 25 patients (73.52%) experienced significant symptomatic relief, and at 4 weeks, 30 (88.23%) recovered and 3 (7.5%) showed improvement. Follow-up at 6 months showed that only 4 (11.76%) of the recovered patients experienced recurrence, which is a much lower recurrence rate than that following antibiotic treatment. There were no adverse effects reported among these cases. RUTI has become a great health problem because of the multidrug and pandrug resistance of the pathogens [4]. The development of new antibiotics and improvements in treatment strategy may alleviate the situation to some extent, but will not change the trend of increasing resistance; however, this can be alleviated and resolved via an alternative treatment strategy. The therapeutic principles and aims of TCM are completely different from that of antibiotic treatment [3]. Instead of focusing on etiologic pathogens, TCM considers the overall functional state of the patient [5]. TCM attempts to restore and improve the functional state disrupted by the pathogen [6]. Therefore, it is impossible for the pathogen to develop resistance to TCM. Here, we indicated that refractory RUTIs can be effectively treated with TCM. Although TCM lacks strict and rigorous clinical trials for many formulas, we believe its extensive use in China for thousands of years has proven its validity [7]. Therefore, to treat refractory RUTI, we suggest that TCM is a good alternative and complementary option.

Multi-drug resistant uropathogenic Escherichia coli and its treatment by Chinese medicine

ObjectivesTo investigate the resistance and virulence profiles of uropathogenic Escherichia coli (UPEC) and its treatment by Chinese medicine (CM) Fuzheng Qingre Lishi Formula (扶正清热利湿方, FQLF).MethodsUPEC strains were isolated from recurrent urinary tract infections (UTIs) patients. Patient sensitivities to 17 antibiotics were tested by the disk diffusion method. Virulence genes were screened by plolymerase chain reaction. A mouse model was constructed using a multi-drug resistant and virulent UPEC strain and treated with FQLF or the antibiotic imipenem. The treatment efficacy was evaluated by bacterial clearance from urine and the urinary organs.ResultsA total of 90 UPEC strains were collected, and 94.4% of the isolates were resistant to at least 1 antibiotic. Approximately 66.7% of the UPEC strains were multi-drug resistant. More than one virulence gene was found in 85.6% of the isolates. The extended-spectrum β-lactamases (ESBL)-positive strains were more resistant than the negative ones. The virulence gene number was positively correlated with the resistance number (P<0.05). A mouse model was successfully constructed using UPEC10. Treatment with either FQLF or antibiotics significantly cleared bacteria from the mouse urine after 14 days. In the untreated control, the bacteria lasted for 28 days. FQLF treatment of the UTI mouse model greatly reduced the bacterial number in the kidney and bladder, but could not completely clear the bacteria.ConclusionsMulti-drug resistance is common among UPEC isolates, and the resistance is positively related with virulence. FQLF could treat UPEC UTIs, but could not completely clear the bacteria from the host.

Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts

Molecular detection assays are increasingly becoming routine diagnostic techniques for bacterial infection; however, their sensitivities are restricted by the low concentrations of bacteria in clinical samples. Here, we report a new paradigm for ultrasensitive detection of bacteria. The principle of this approach is that by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts, assay sensitivity can be greatly improved. First, signature genes with abundant transcripts were screened by RNA-Seq. We confirmed that RT-PCR efficiently amplifies both DNA and RNA, while PCR amplifies only DNA. Unexpectedly, we found that the RNA extraction efficiency is relatively low, while simplified denaturation was more appropriate for transcript detection. For highly transcribed genes, RT-PCR consistently generated lower cycle threshold (Ct) values than those of PCR. The sensitivity of RT-PCR targeting abundant transcripts could detect quantities as low as one bacterium, which was not possible using PCR. Amplification of different genes among several other common bacteria also confirmed that transcript detection by RT-PCR is more sensitive than is DNA detection by PCR. Therefore, abundant transcript detection represents a universal strategy for ultrasensitive detection of bacteria.

Recurrent urinary tract infections caused by multidrug-resistant uropathogenic Escherichia coli: implications for diagnosis and treatment.

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