Wenli Zhang

Specific patterns of gyrA mutations determine the resistance difference to ciprofloxacin and levofloxacin in Klebsiella pneumoniae and Escherichia coli

BackgroundWide use of ciprofloxacin and levofloxacin has often led to increased resistance. The resistance rate to these two agents varies in different clinical isolates of Enterobacteriaceae. Mutations of GyrA within the quinolone resistance-determining regions have been found to be the main mechanism for quinolone resistance in Enterobacteriaceae. It has been shown that only some of the mutations in the gyrA gene identified from clinical sources were involved in fluoroquinolone resistance. Whether different patterns of gyrA mutation are related to antimicrobial resistance against ciprofloxacin and levofloxacin is unclear.MethodsThe minimum inhibitory concentration (MIC) of ciprofloxacin and levofloxacin were determined by the agar dilution method followed by PCR amplification and sequencing of the quinolone resistance determining region of gyrA to identify all the mutation types. The correlation between fluoroquinolone resistance and the individual mutation type was analyzed.ResultsResistance differences between ciprofloxacin and levofloxacin were found in 327 isolates of K. pneumoniae and E. coli in Harbin, China and in the isolates reported in PubMed publications. GyrA mutations were found in both susceptible and resistant isolates. For the isolates with QRDR mutations, the resistance rates to ciprofloxacin and levofloxacin were also statistically different. Among the 14 patterns of alterations, two single mutations (Ser83Tyr and Ser83Ile), and three double mutations (Ser83Leu+Asp87Asn, Ser83Leu+Asp87Tyr and Ser83Phe+Asp87Asn) were associated with both ciprofloxacin and levofloxacin resistance. Two single mutations (Ser83Phe and Ser83Leu) were related with ciprofloxacin resistance but not to levofloxacin. Resistance difference between ciprofloxacin and levofloxacin in isolates harboring mutation Ser83Leu+Asp87Asn were of statistical significance among all Enterobacteriaceae (P<0.001).ConclusionsResistance rate to ciprofloxacin and levofloxacin were statistically different among clinical isolates of Enterobacteriaceae harboring GyrA mutations. Ser83Leu+Asp87Asn may account for the antimicrobial resistance difference between ciprofloxacin and levofloxacin.

Alteration of GyrA Amino Acid Required for Ciprofloxacin Resistance in Klebsiella pneumoniae Isolates in China

ABSTRACT Resistance to ciprofloxacin was detected in 111 (48.1%) isolates of Klebsiella pneumoniae from China. GyrA alterations were identified in the ciprofloxacin-resistant and ciprofloxacin-susceptible isolates. The results, including previously published data, indicate that the single substitution Ser83→Ile and three types of double mutations at Ser83 and Asp87 were required for ciprofloxacin resistance (P < 0.05).

Multilocus sequence types and virulence determinants of hypermucoviscosity-positive Klebsiella pneumoniae isolated from community-acquired infection cases in Harbin, North China

We investigated the molecular epidemiologic characteristics and virulence of hypermucoviscosity-positive Klebsiella pneumoniae in mainland China. We detected 16 hypermucoviscosity-positive strains in 65 total clinical isolates (24.62%). We found that 68.75% (11/16) of the positive strains had K2 genotype and carried the rmpA and iucA genes. Multilocus sequence typing revealed 5 sequence types (STs): ST65 [7], ST23 [4], ST86 [3], ST412 [1], ST375 [1], whereas the remaining 4 isolates were defined as other STs. The order of the median lethal dose values for the ST types was ST23 (2.19 × 10(3) CFU/mouse) < ST86 (1.70 × 10(4) CFU/mouse) < ST65 (5.05 × 10(7) CFU/mouse) < the other STs (1.90 × 10(8) CFU/mouse). In conclusion, the K2 with ST65 carrying rmpA and iucA was the most predominant among the hypermucoviscosity-positive K. pneumoniae strains obtained from community-acquired infection cases in Harbin, North China. Sequence types are a valuable tool to predict the risk of K. pneumoniae infection.

Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains

Resistance to β-lactam antibiotics through β-lactamase production by Enterobacteriaceae continues to burden the health-care sector worldwide. Traditional methods for detection of β-lactamases are time-consuming and labor-intensive and newer methods with varying capabilities continue to be developed. The objective of this study was to develop a multiplex PCR (M-PCR) system for the detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes and to apply it in clinical Klebsiella pneumoniae and Escherichia coli strains. To do this, we used group-specific PCR primers in singleplex reactions followed by optimization into multiplex reactions. Specificity and sensitivity of the M-PCR were then evaluated using 58 reference strains before its application to detect bla group genes in 203 clinical Enterobacteriaceae strains. PCR amplicons were sequenced to determine the β-lactamase subtypes. The M-PCR system exhibited 100% specificity and sensitivity. In all, 83.7% of K. pneumoniae and 89.8% of E. coli clinical strains harbored bla group genes with 46.9%, 40.1%, 15.0%, 21.1% and 6.1% of K. pneumoniae having blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively, whereas 12.2%, 77.6%, 22.4%, 36.7% and 8.2% of E. coli had blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively. BlaSHV-1, blaSHV-11, blaSHV-27, blaSHV-33, blaSHV-144, blaTEM-1, blaTEM-135, blaOXA-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, blaCTX-M-65 and blaCTX-M-104 were detected. In conclusion, the M-PCR system was efficient and versatile with an advantage of simultaneously detecting all the targeted bla group genes. Hence, it is a potential candidate for developing M-PCR kits for the screening of these genes for clinical or epidemiological purposes.

Comparative Analysis of Quinolone Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli from Chinese Children and Adults

The objective of this study was to compare quinolone resistance and gyrA mutations in clinical isolates of Klebsiella pneumoniae and Escherichia coli from Chinese adults who used quinolone in the preceding month and children without any known history of quinolone administration. The antimicrobial susceptibilities of 61 isolates from children and 79 isolates from adults were determined. The mutations in the quinolone resistance-determining regions in gyrA gene were detected by PCR and DNA sequencing. Fluoroquinolone resistance and types of gyrA mutations in isolates from children and adults were compared and statistically analyzed. No significant differences were detected in the resistance rates of ciprofloxacin and levofloxacin between children and adults among isolates of the two species (all P > 0.05). The double mutation Ser83→Leu + Asp87→Asn in the ciprofloxacin-resistant isolates occurred in 73.7% isolates from the children and 67.9% from the adults, respectively (P = 0.5444). Children with no known history of quinolone administration were found to carry fluoroquinolone-resistant Enterobacteriaceae isolates. The occurrence of ciprofloxacin resistance and the major types of gyrA mutations in the isolates from the children were similar to those from adults. The results indicate that precautions should be taken on environmental issues resulting from widespread transmission of quinolone resistance.

The bla CTX-M gene independently enhances drug resistance level to ampicillin in clinical isolates of Klebsiella pneumoniae

The bla CTX-M gene independently enhances drug resistance level to ampicillin in clinical isolates of Klebsiella pneumoniae

Detection of mycobacterial small RNA in the bacterial culture supernatant and plasma of patients with active tuberculosis

Bacterial small RNA (sRNA) has been shown to play an important role in control of bacteria virulence, stress response and physiological metabolism by post-transcriptional regulation of gene expression. However, there were few reports about bacterial sRNA as a biomarker of infection. To test the potential role of sRNA in indicating infection of Mycobacterium tuberculosis, total RNA were extracted from the filtrated bacterial cultural supernatant. After synthesis of cDNA by reverse transcription, four Mycobacterial sRNAs including ASdes, ASpks, AS1726, and AS1890, which have been experimentally confirmed by Kristine B in the year of 2009, were detected by real time PCR. The specificity was verified by sequencing of the amplified products. Moreover, we demonstrate that the presence of sRNA Asdes in plasma of 55.56% (15/27) TB patients and 25.00% (6/24) normal controls with BCG vaccination (P < 0.05). Our results suggest that bacterial non-coding sRNA can be detected from either bacterial culture supernatants or patients plasma. Detecting of Mycobacterial sRNA provides a rapid and relatively noninvasive approach for diagnosing disease and could be developed as a biomarker to identify patients with active tuberculosis to help make informed decisions about proper therapies.1.