Characterization of blaAFM-1-positive carbapenem-resistant strains isolated in Guangzhou, China.

BACKGROUND: Carbapenemase-producing makes a great contribution to carbapenem resistance in Gram-negative bacilli. BlaAFM-1 gene was first discovered by us in Alcaligenes faecalis AN70 strain isolated in Guangzhou of China and, was submitted to NCBI on 16 November 2018. METHODS: Antimicrobial susceptibility testing was performed by broth microdilution assay using BD Phoenix 100. The phylogenetic tree of AFM and other B1 metallo-ß-lactamases was visualized by MEGA7.0. Whole-genome sequencing technology was used to sequence carbapenem-resistant strains including the blaAFM-1 gene. Cloning and expressing of blaAFM-1 were designed to verify the function of AFM-1 to hydrolyze carbapenems and common ß-lactamase substrates. Carba NP and Etest experiments were conducted to evaluate the activity of carbapenemase. Homology modeling was applied to predict the spatial structure of AFM-1. A conjugation assay was performed to test the ability of horizontal transfer of AFM-1 enzyme. The genetic context of blaAFM-1 was performed by Blast alignment. RESULTS: Alcaligenes faecalis strain AN70, Comamonas testosteroni strain NFYY023, Bordetella trematum strain E202, and Stenotrophomonas maltophilia strain NCTC10498 were identified as carrying the blaAFM-1 gene. All of these four strains were carbapenem-resistant strains. Phylogenetic analysis revealed that AFM-1 shares little nucleotide and amino acid identity with other class B carbapenemases (the highest identity (86%) with NDM-1 at the amino acid sequence level). The spatial structure of the AFM-1 enzyme was predicted to be αß/ßα sandwich structure, with two zinc atoms at its active site structure. Cloning and expressing of blaAFM-1 verified AFM-1 could hydrolyze carbapenems and common ß-lactamase substrates. Carba NP test presented that the AFM-1 enzyme possesses carbapenemase activity. The successful transfer of pAN70-1(plasmid of AN70) to E.coli J53 suggested that the blaAFM-1 gene could be disseminated by the plasmid. The genetic context of blaAFM indicated that the downstream of the blaAFM gene was always adjacent to trpF and bleMBL. Comparative genome analysis revealed that blaAFM appeared to have been mobilized by an ISCR27-related mediated event. CONCLUSIONS: The blaAFM-1 gene is derived from chromosome and plasmid, and the blaAFM-1 gene derived from the pAN70-1 plasmid can transfer carbapenem resistance to susceptible strains through horizontal transfer. Several blaAFM-1-positive species have been isolated from feces in Guangzhou, China.

Selective Photothermal Therapy Based on Lipopolysaccharide Aptamer Functionalized MoS2 Nanosheet-Coated Gold Nanorods for Multidrug-Resistant Pseudomonas aeruginosa Infection.

Chronic wounds infected by multidrug-resistant gram-negative bacteria have evolved resistance to traditional antibiotic therapy, posing a threat to global public health in recent years. Herein, a selective therapeutic nanorod (MoS2 -AuNRs-apt) based on molybdenum disulfide (MoS2 ) nanosheets coated gold nanorods (AuNRs) targeting lipopolysaccharide (LPS) is presented. AuNRs have excellent photothermal conversion efficiency in 808 nm laser-guided photothermal therapy (PTT), and the MoS2 nanosheets coating significantly enhances the biocompatibility of AuNRs. Furthermore, the conjugation of the nanorods with aptamer permits active targeting of LPS on the surface of gram-negative bacteria and a specific anti-inflammatory ability in the multidrug-resistant Pseudomonas aeruginosa (MRPA)-infected wound murine model. It is concluded that the antimicrobial effect of these nanorods is considerably more significant than non-targeted PTT. Moreover, they can precisely overcome MRPA bacteria by physical damage and effectively reduce excess M1 inflammatory macrophages to accelerate the healing of infected wounds. Overall, this molecular therapeutic strategy displays great potential as a prospective antimicrobial treatment for MRPA infections.

Rapid detection for infected ascites in cirrhosis using metagenome next-generation sequencing: A case series.

Empirical antibiotic therapy in patients with spontaneous bacterial peritonitis (SBP) is common as pathogen(s) are identified in only 5%-20% patients using conventional culture-based techniques. Metagenome next-generation sequencing (mNGS) test is a promising approach for the diagnosis of infectious disease. The clinical application of mNGS for infected ascites in cirrhotic patients is rarely reported. Here, we describe three cases to preliminarily explore the potential role of mNGS for microbiological diagnosis of ascites infection in an exploratory manner. The clinical performance of ascites mNGS in cirrhotic patients remains to be further evaluated.

Rapid detection of a novel B1-ß-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay.

BACKGROUND: BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-ß-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection. METHODS: We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains. RESULTS: The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8-100%) and 100% specificity (95% CI 98.4-100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection. CONCLUSIONS: We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-ß-lactamase gene, blaAFM-1.

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2.

BACKGROUND: The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease 2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56°C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. METHODS: By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56°C for 30 minutes using a quantitative fluorescence immunochromatographic assay RESULTS: We showed that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 disease group (n = 9). Of note, 44.12% of the detected IgM levels were dropped below the cutoff value after heating, suggesting heat inactivation can lead to false-negative results of these samples. CONCLUSION: Our results indicate that heat inactivation of serum at 56°C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended, and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.

Nanoparticles for the Treatment of Oral Biofilms: Current State, Mechanisms, Influencing Factors, and Prospects.

Due to their excellent size, designability, and outstanding targeted antibacterial effects, nanoparticles have become a potential option for controlling oral biofilm-related infections. However, the formation of an oral biofilm is a dynamic process, and factors affecting the performance of antibiofilm treatments are complex. As such, when examining the existing literature on the antibiofilm effects of nanoparticles, attention should be paid to the specific mechanisms of action at different stages of oral biofilm formation, as well as relevant influencing factors, in order to achieve an objective and comprehensive evaluation. This review is intended to detail the antibacterial mechanisms of nanoparticles during the four stages of the formation of oral biofilms: 1) acquired film formation; 2) bacterial adhesion; 3) early biofilm development; and 4) biofilm maturation. In addition, factors influencing the antibiofilm properties of nanoparticles are summarized from the aspects of nanoparticles themselves, biofilm models, and host factors. The limitations of current research and possible trends for future research are also discussed. In summary, nanoparticles are a promising antioral biofilm strategy. It is hoped that this review can serve as a reference and inspire ideas for further research on the application of nanoparticles for effectively targeting and treating oral biofilms.

Molecular characteristics of carbapenem-resistant Acinetobacter spp. from clinical infection samples and fecal survey samples in Southern China.

BACKGROUND: Carbapenem resistance among Acinetobacter species has become a life-threatening problem. As a last resort in the treatment of gram-negative bacteria infection, resistance to colistin is also a serious problem. The aim of study was to analyze the mechanism of resistance and perform genotyping of carbapenem-resistant Acinetobacter from clinical infection and fecal survey samples in Southern China. METHODS: One hundred seventy and 74 carbapenem-resistant Acinetobacter were isolated from clinical infection samples and fecal survey samples, respectively. We detected the related genes, including carbapenemase genes (blaKPC, blaIMP, blaSPM, blaVIM, blaNDM, blaOXA-23-like, blaOXA-24/40-like, blaOXA-51-like, and blaOXA-58-like), colistin resistance-related genes (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5), a porin gene (carO), efflux pump genes (adeA, adeB, adeC, adeI, adeJ, and adeK), mobile genetic element genes (intI1, intI2, intI3, tnpU, tnp513, IS26, ISAba1, and ISAba125), and the integron variable region. Genotyping was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and dendrogram cluster analysis. RESULTS: Among the 244 carbapenem-resistant Acinetobacter, the common carbapenemase-positive genes included the following: blaOXA-51-like, 183 (75.00%); blaOXA-23-like, 174 (71.30%); blaNDM-1, 57 (23.40%); and blaOXA-58-like, 30 (12.30%). The coexistence of mcr-1 and blaNDM-1 in five strains of A. junii was found for the first time. Eleven distinct carO gene variants were detected in 164 (67.20%) strains, and ten novel variants, which shared 92-99% identity with sequences in the Genbank database, were first reported. Efflux system genes were present in approximately 70% of the isolates; adeABC and adeIJK were observed in 76.23 and 72.13%, respectively. Class 1 integrons were detected in 180 (73.80%) strains and revealed that four gene cassette arrays contained 11 distinct genes. The genotyping by ERIC-PCR demonstrated a high genetic diversity of non-baumannii Acinetobacter, and greater than 90% similarity to A. baumannii. CONCLUSIONS: The blaNDM-1 gene was identified in up to 77% of the carbapenem-resistant Acinetobacter isolated from fecal survey samples, indicating that the gut might be a reservoir of resistant opportunistic bacteria. Intestinal bacteria can be transmitted through the fecal-hand, which is a clinical threat, thus, the monitoring of carbapenem-resistant bacteria from inpatients' feces should be improved, especially for patients who have been using antibiotics for a long time.

Multiplex real-time PCR assays to detect Stenotrophomonas maltophilia carrying sul1, sul2, and sul3 genes.

Nosocomial infections caused by Stenotrophomonas maltophilia resistant to SXT are increasingly reported worldwide. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of the ssrA and sul1, sul2 and sul3 genes was first established. The assays were performed on a Roche LightCycler® 480 II system. The results for target and non-target amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 µL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were <5%. The sensitivity for the target DNA in simulated blood samples was 102 CFU/mL. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 20 SXT-susceptible and 20 SXT-resistant S. maltophilia from clinical samples. Therefore, the multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of the ssrA and sul1, sul2 and sul3 genes.

Detection of common mobile genetic elements and genotyping of multidrug-resistant Gram-negative bacilli in blood specimens from septicemia patients in southern China.

BACKGROUND: Integron, ISCR1 and complex class 1 integrons lead bacteria to become resistant to antibiotic regimens. The aim of this study was to detect common mobile genetic elements of multidrug-resistant Gram-negative bacilli and evaluate the genotyping of these bacilli in blood specimens from septicemia patients in southern China. METHODS: A total of 837 Gram-negative bacilli including 578 strains containing Enterobacteriaceae and 259 strains containing non-fermentative bacilli were investigated in blood samples collected from septicemia patients between 2011 and 2014 in southern China. Mobile genetic elements, such as class 1 integrons, the insertion sequence common region 1 (ISCR1), and complex class 1 integrons, were detected from the 837 strains. RESULTS: Twenty-seven types of gene cassette arrays were found among 837 strains in which 492 (58.8%) class 1 integron-positive isolates and 254 (51.6%) gene cassette-positive isolates were found, including the first description of two types, aacA4-bla IMP-1-bla OXA-30-catB3 and aac(6')-II-aadA13-cmlA8-bla OXA-10, in the corresponding species and two gene cassettes, putative helicase and aadA-like, originally detected in integrons. Twelve types of ISCR1-linked resistance gene regions in 196 ISCR1-positive bacilli and seven different types of complex class 1 integron-positive strains were obtained including four distinct complex class 1 integrons that have never been described in any species. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fngerprinting showed that isolates with identical gene profles were clonally unrelated. CONCLUSION: Our results indicated that we should pay more attention to enhance the quality of infection control measures and prevent hospital infection, so as to avoid the outbreak of multidrug-resistant Gram-negative bacilli.

Integrons and insertion sequence common region 1 (ISCR1) of carbapenem-non-susceptible Gram-negative bacilli in fecal specimens from 5000 patients in southern China.

The integrons and insertion sequence common region 1 (ISCR1) of 329 carbapenem-non-susceptible Gram-negative bacilli, excluding 60 Stenotrophomonas maltophilia strains, in fecal specimens from 5000 patients in southern China were studied. A total of 205 (62.3%) class 1 integron-positive strains and 126 (61.5%) gene-cassette-positive strains were detected, and 44 types of gene-cassette arrays were found, including 8 types described for the first time in the corresponding species and one gene cassette originally detected in integrons, a putative helicase. A total of 9 types of 21 ISCR1-linked resistance gene regions in 35 ISCR1-positive bacilli and 2 previously unreported types of complex class 1 integrons were detected. All strains were extensively drug-resistant. Class 1 integrons and ISCR1 were widely disseminated and these structures carried various resistance genes, which were closely associated with mechanisms of multidrug resistance, including carbapenem resistance.

Two Multiplex Real-Time PCR Assays to Detect and Differentiate Acinetobacter baumannii and Non- baumannii Acinetobacter spp. Carrying blaNDM, blaOXA-23-Like, blaOXA-40-Like, blaOXA-51-Like, and blaOXA-58-Like Genes.

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S-23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 µL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections.

New structures simultaneously harboring class 1 integron and ISCR1-linked resistance genes in multidrug-resistant Gram-negative bacteria.

BACKGROUND: The connection structure of class 1 integron and insertion sequence common region 1 (ISCR1) is called "complex class 1 integrons" or "complex sul1-type integrons", which is also known to be associated with many resistance genes. This structure is a powerful gene-capturing tool kit that can mobilize antibiotic resistance genes. In order to look for and study the structure among clinical multidrug-resistant (MDR) Gram-negative isolates, 63 isolates simultaneously harbored class 1 integron and ISCR1-linked resistance genes were isolated from 2309 clinical non-redundant MDR Gram-negative isolates in Nanfang Hospital in 2008-2013. The connecting regions between the class 1 integrons and ISCR1 were examined using PCR and DNA sequencing to determine the structures in these isolates. RESULT: The two elements (the variable regions of the class 1 integron structures and the ISCR1-linked resistance genes) are connected in series among 63 isolates according to long-extension PCR and DNA sequencing. According to the kinds and permutations of resistance genes in the structure, 12 distinct types were identified, including 8 types that have never been described in any species. Several types of these structures are similar with the structures of other reports, but not entirely same. CONCLUSION: This study is the first to determine the structure simultaneously harboring class 1 integron and ISCR1-linked resistance genes by detecting the region connecting class 1 integrons and ISCR1 in a large number of MDR bacteria. These structures carrying various resistance genes were closely associated with multidrug resistance bacteria in Southern China.

Molecular characteristics of carbapenem-resistant gram-negative bacteria in southern China.

A total of 368 nonreplicate gram-negative bacteria with resistance to imipenem or meropenem were collected to search for carbapenemase genes, class 1 integrons, and insertion sequence with common region 1 (ISCR1). The carbapenemase genes blaIMP-4, blaKPC-2, and blaNDM-1 were found in two Enterobacteriaceae and seven Pseudomonas aeruginosa isolates, nine Klebsiella pneumoniae isolates, and seven Enterobacteriaceae and two Acinetobacter spp. isolates. The class D OXA-type carbapenemase genes blaOXA-23-like, blaOXA-24-like, blaOXA-58, and blaOXA-51-like were detected in 59 (34.9%), 2 (1.2%), 16 (9.5%), and 126 (74.6%) Acinetobacter strains. This is the first description of blaNDM-1 in Enterobacter hormaechei and Acinetobacter genomic species 13TU. Of the integrase-positive strains, 135 (90.0%) Acinetobacter spp., 22 (61.1%) P. aeruginosa, and 14 (100%) Enterobacteriaceae isolates were identified by five, ten, and four different gene cassette arrays, respectively. Three novel gene cassette arrays aadB-aadA1, dfrA25, and dfrA16-aadA2 were reported for the first time in some species. Of the ISCR1-positive strains, the nonfermentative strains (102 Acinetobacter spp. and 13 P. aeruginosa. isolates) contained the same arrangement blaPER-1-putative glutathione-S-transferase-novel type ABC transporter, and three Enterobacteriaceae isolates harbored three different arrangements. Four distinct complex class 1 integron structures were observed. The complex class 1 integron detected in New Delhi, metallo-ß-lactamase (NDM-1)-producing E. hormaechei, was found to coexist in the NDM-1-carrying plasmid. Our results suggested that we should pay more attention to the strict implementation of infection control measures and active antibiotic resistance surveillance to avoid the rapid spread or outbreak of carbapenemase-producing gram-negative bacteria.

[Combined drug sensitivity test of 50 strains of extensively drug-resistant Acinetobacter baumannii].

OBJECTIVE: To study the in vitro antibacterial activity of meropenem combined with doxycycline, ciprofloxacin, sulbactam or cefoperazone/sulbactam against clinically isolated extensively drug-resistant Acinetobacter baumannii (XDRAB). METHODS: Using a checker board synergy design, the minimal inhibitory concentration (MIC) of antibiotics against 50 isolates of XDRAB was determined by broth microdilution antifungal susceptibility test. The fractional inhibitory concentration (FIC) index was calculated to determine the combined effect of the antibiotics. RESULTS: Meropenem showed significantly reduced MIC50 and enhanced antimicrobial activities when combined with doxycycline, sulbactam or cefoperazone/sulbactam. The FIC results suggested that the main actions of doxycycline, sulbactam, and cefoperazone/sulbactam were synergistic (38%, 26%, and 10%, respectively) and addictive (62%, 74%, and 90%, respectively) without indifferent or antagonistic effects. The main actions of meropenem combined with ciprofloxacin were additive (56%) and indifference (44%) with synergistic and antagonistic effects. CONCLUSION: Meropenem combined with doxycycline, sulbactam or cefoperazone/sulbactam shows excellent activity against clinical isolates of XDRAB.

Rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of blaKPC, blaNDM, blaIMP, and blaVIM carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with blaNDM, 11 strains with blaKPC, 11 strains with blaVIM, and 2 strains with blaIMP obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria.

Construction and characterization of a Vibrio cholerae serogroup O139 vaccine candidate by genetic engineering.

The present study aimed to construct and evaluate the live attenuated Vibrio cholerae serogroup O139 vaccine candidate, in which genes encoding protective antigens were integrated into the chromosomal DNA. Using the initial strain, O139-ZJ9693, the toxin-linked cryptic (TLC) and cholera toxin (CTX) genetic elements and repeats in the toxin (RTX) gene cluster were deleted from its chromosomal DNA, and the cholera toxin genes, ctxB and rstR, were transferred into the chromosome to construct the candidate vaccine strain. The expression of ctxB and the vaccine virulence were then examined. Polymerase chain reaction (PCR), enzymatic digestion and electrophoresis were performed to confirm that TLC, CTX and RTX were deleted, and that ctxB and rstR were transferred into the vaccine candidate DNA. According to the preliminary evaluation, the ctxB gene exhibited cholera toxin subunit B expression, and no enterotoxigenic or cytotoxic effects were observed in this strain. In conclusion, a recombinant strain containing genes encoding protective antigens that replaced virulence-associated genes was successfully constructed in the present study; this candidate strain may have the potential to be utilized to further evaluate the immune response.

Molecular identification of clinical "difficult-to-identify" microbes from sequencing 16S ribosomal DNA and internal transcribed spacer 2.

BACKGROUND: Clinical microbiology laboratories have to accurately identify clinical microbes. However, some isolates are difficult to identify by the automated biochemical text platforms, which are called "difficult-to-identify" microbes in this study. Therefore, the ability of 16S ribosomal DNA (16S rDNA) and internal transcribed spacer 2 (ITS2) sequencing to identify these "difficult-to-identify" bacteria and fungi was assessed in this study. METHODS: Samples obtained from a teaching hospital over the past three years were examined. The 16S rDNA of four standard strains, 18 clinical common isolates, and 47 "difficult-to-identify" clinical bacteria were amplified by PCR and sequenced. The ITS2 of eight standard strains and 31 "difficult-to-identify" clinical fungi were also amplified by PCR and sequenced. The sequences of 16S rDNA and ITS2 were compared to reference data available in GenBank by using the BLASTN program. These microbes were identified according to the percentage of similarity to reference sequences of strains in GenBank. RESULTS: The results from molecular sequencing methods correlated well with automated microbiological identification systems for common clinical isolates. Sequencing results of the standard strains were consistent with their known phenotype. Overall, 47 "difficult-to-identify" clinical bacteria were identified as 35 genera or species by sequence analysis (with 10 of these identified isolates first reported in clinical specimens in China and two first identified in the international literature). 31 "difficult-to-identify" clinical fungi tested could be identified as 15 genera or species by sequence analysis (with two of these first reported in China). CONCLUSIONS: Our results show the importance of 16S rDNA and internal ITS2 sequencing for the molecular identification of "difficult-to-identify" bacteria and fungi. The development of this method with advantages of convenience, availability, and cost-effectiveness will make it worth extending into clinical practice in developing countries.

The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in bacteria.

The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-ß-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of blaNDM and blaKPC with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for blaNDM (R2 = 0.971; slope, -3.273) and blaKPC (R2 = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.

Class 1 integrons in urinary isolates of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Southern China during the past five years.

We analyzed extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (226) and Klebsiella pneumoniae (53) collected from urine specimens during 2005-2009 for the presence of ESBL genes, class 1 integrons, and characterization of gene cassettes. TEM and CTX-M ß-lactamase genes were the most prevalent. One hundred and forty-four E. coli and 35 K. pneumoniae were positive for the class 1 integrase gene; among them, 99 E. coli and 14 K. pneumoniae detected gene cassettes. Gene cassette regions were identified by restriction fragment length polymorphism and DNA sequencing analysis. Eleven distinct gene cassette arrays were found in E. coli isolates, and seven distinct gene cassette arrays were found in K. pneumoniae isolates. The cassette array aacA4-catB8-aadA1 was first found in E. coli and dfrA1-orfC was first reported in K. pneumoniae. Most of the gene cassettes found in this class 1 integrons were for aminoglycoside resistance. Enterobacterial repetitive intergenic consensus-polymerase chain reaction fingerprint patterns revealed the isolates carrying gene cassettes were genetically unrelated. In conclusion, we studied the class 1 integrons among urinary isolates of ESBL-producing E. coli and K. pneumoniae in Southern China during the past 5 years and found that class 1 integrons were widely disseminated and played a major role in antibiotic resistance.

Novel ISCR1-linked resistance genes found in multidrug-resistant Gram-negative bacteria in southern China.

Non-duplicate multidrug-resistant (MDR) Gram-negative bacteria (n=1329) isolated from southern China between January 2008 and December 2009 were investigated for the presence of ISCR1 as well as characterisation of ISCR1-linked resistance genes. Of 433 ISCR1-positive strains, 151 appeared to carry ISCR1-linked resistance genes. Seven different ISCR1-linked resistance gene arrays were identified by restriction fragment length polymorphism (RFLP) and DNA sequencing analysis. Many of these arrays are reported in some species for the first time. A total of 12 genes, including a novel ABC transporter (GenBank accession no. GU944725), qnrA1, qnrB2, qnrB6, bla(DHA-1), ampR, bla(CTX-M-9), bla(PER-1), insB, sapA-like peptide transport periplasmic protein, putative glutathione S-transferase and short-chain dehydrogenase/reductase, were detected. This study was the first to employ PCR-RFLP using HinfI and RsaI to analyse ISCR1-linked genes. ISCR1 was widely disseminated among MDR Gram-negative bacteria and was in close association with quinolone resistance and ß-lactamase genes (class A and class C) in southern China.