[Emergence of novel variants of gyrA, parC, qnrS genes in multi-drug resistant Klebsiella caused pneumonia].

OBJECTIVE: To investigate the resistant mechanism of quinolones on multi-drug resistant Klebsiella caused pneumonia (MDR-KPN). METHODS: From August 2008 to May 2010, 47 strains of MDR-KPN were collected from 6 hospitals in Hangzhou and Huzhou in Zhejiang province in China. Drug target genes to quinolones (gyrA, parC) and quinolone-resistance genes mediated by mobile genetic elements [qnrA, qnrB, qnrS, aac (6')-Ib-cr, qepA] were analyzed by PCR and verified by DNA sequencing. RESULTS: Positive results were found in 47 strains of MDR-KPN, 43 strains (91.5%) of gyrA mutation, 40 strains (85.1%) of parC mutation, 3 strains (6.4%) of qnrB2, 1 strain (2.1%) of qnrB 4, 8 strains (17.0%) of qnrS 1, 5 strains (10.6%) of qnrS 4, 2 strains (4.3%) of aac (6')-Ib-cr respectively. Moreover, 5 novel variants of gyrA (GenBank accession number: JN811952, JN811953, JN811954, JN811955, JN811956), 5 novel variants of parC (GenBank accession number: JN817432, JN817433, JN817434, JN817435, JN817436) were also identified. In addition, qnrS4 (GenBank accession number: JN836269) appeared to be the novel variants of qnrS. CONCLUSION: Quinolone-resistance-determining region played a key role on the resistance to quinolones in this group of MDR-KPN, and quinolone-resistance genes mediated by mobile genetic elements [qnrB2, qnrB4, qnrS1, qnrS4, aac (6')-Ib-cr] showed positive in some parts of the strains. This was the first report on emergence of qnrS4 in the world.

[Study on pan-resistant Klebsiella pneumoniae harboring blaKPC-2 type carbapenemase gene from a hospital outbreak in Huzhou, Zhejiang].

OBJECTIVE: To investigate the status of genotype of the KPC (Klebsiella pneumoniae carbapenemase)-encoding genes in Pan-resistant K. Pneumoniae, isolated from the 98th Hospital of People's Liberation Army, Huzhou district, Zhejiang province, China. METHODS: 19 strains of Pan-resistant K. pneumoniae were isolated from the inpatients between November, 2008 and July, 2009. Phenotypic confirmatory test for suspected carbapenemases production were carried out by Modified Hodge test. Carbapenemase gene of bla(KPC) was analyzed by PCR and verified by DNA sequencing. RESULTS: In 19 strains of K. pneumoniae, the positive rates of Modified Hodge test and gene of bla(KPC) were both 100.0%. These genes all belonged to bla(KPC-2) subtype confirmed by nucleotide sequence analysis. Among them, the bla(KPC-2) gene sequence of the HZ001 strain (its original serial number was HZ9871) had been registered in GenBank (GenBank Accession Number: GU086225). CONCLUSION: All of the Pan-resistant K. pneumoniae isolated from the inpatients harbored bla(KPC-2) type carbapenemases gene and causing an outbreak in a hospital. Carbapenemases that producing type KPC-2 might be the major reason which causing the resistance to Carbapenems antibiotics.

[Analysis on 168 rRNA methylase genes and aminoglycoside modifying enzymes genes in Enterobacter cloacae in China].

OBJECTIVE: To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes (AMEs) genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital, Huzhou district, Zhejiang province, China. METHODS: 40 strains of Enterobacter cloacae were isolated from the inpatients between September, 2003 and November, 2004. 5 kinds of 16S rRNA methylase gene (including armA, rmtA, rmtB, rmtC and rmtD) and 9 kinds of AMEs gene [including aac (3)-I, aac(3)-II, aac(3)-III, aac(3)-IV, aac(6')-Ib, aac(6')-II, ant(3")-I, ant(2")-I and aph(3')-VI] were analyzed by PCR and verificated by DNA sequencing. RESULTS: In 40 strains of Enterobacter cloacae, the positive rates of genes of rmtB, aac(3)-II, aac(6')-I b, ant(3")-I, ant(2")-I and aph(3')-VI were 12.5% (5/40), 27.5% (11/40), 72.5% (29/40), 32.5% (13/40), 5.0% (2/40) and 5.0% (2/40), respectively. 8 kinds of the rest of genes were all tested negative. The total positive rate of AMEs gene was 85.0% (34/40). Among 29 strains of Enterobacter cloacae that the aac (6')-I b gene was positive, through PCR and verification by DNA sequencing, 7 strains (24.1%) were confirmed to take the aac(6')-I b-cr (the GenBank register number: EF375620, EU159121) alone, 18 strains (62.1%) were confirmed to take the aac(6')-I b-Suzhou (EU085533) alone, 3 strains (10.3%) were confirmed to take both aac(6')-I b-Suzhou and aac(6')-I b-cr while only 1 (3.4%) was aac(6')-I b (the classical type). CONCLUSION: There was lower positive rate of 16S rRNA methylase gene but very high AMEs genotypes in Enterobacter cloacae isolated from inpatients and the finding of rmtB gene was reported for the first time in the world. At least 5 kinds of AMEs gene existed in Enterobacter cloacae were isolated and they were the new host of both gene of aac(6')-I b- cr and aac(6')-I b-Suzhou, with aac(6')-I b-Suzhou gene was the predominance subtype in aac(6')-I b.

[Emergence of 16S rRNA methylase gene rmtB in Klebsiella pneumoniae isolates from the inpatients in Zhejiang Province, China].

OBJECTIVE: To investigate the presence and genetic background of 16S rRNA methylase gene and Aminoglycoside modifying enzymes (AMEs) genes in Klebsiella pneumoniae isolated from the People's Liberation Army 98th Hospital, Huzhou district, Zhejiang province, China. METHODS: 25 strains of Klebsiella pneumoniae were isolated from the inpatients between September, 2005 and April, 2006. 6 kinds of 16S rRNA methylase gene (including armA, rmtA, rmtB, rmtC, rmtD and npmA), 6 kinds of AMEs genes [including aac (3)-I, aac (3)-II, aac (6')-I, aac (6')-II, ant (3")-I and ant (2")-I], intI1, intI2, intI3, mercuric reductase gene merA (merA gene were the collective genetic markers of transposons of Tn21 and Tn501) and tnpA (tnpA gene were the collective genetic markers of transposons of Tn1, Tn2,Tn3 and Tn1000) were analyzed by PCR and verificated by DNA sequencing. RESULTS: In 25 strains of Klebsiella pneumoniae, the positive rate of genes of rmtB, aac (3)-II, aac (6')-I, ant (3")-I and intI1 were 60.0% (15/25), 4.0% (1/25), 48.0% (12/25), 60.0% (15/25) and 96.0% (24/25), respectively. The rest 12 kinds of genes were all tested negative. The total positive rate of 6 kinds of AMEs gene was 84.0% (21/25). CONCLUSION: There were very high positive rate on both genes of rmtB and AMEs genotypes in Klebsiella pneumoniae isolated from inpatients, and this was the first report of the emergence of 16S rRNA methylase gene rmtB in Klebsiella pneumoniae identified in Zhejiang province, China.

[Study on the molecular epidemiology of SHV type beta-lactamase-encoding genes of multiple-drug-resistant acinetobacter baumannii].

OBJECTIVE: To analyse the plasmid-mediated SHV type beta-lactamases-encoding genes sequence and to identify its subtype of multiple-drug-resistant acinetobacter baumannii isolated from Huzhou district, Zhejiang province, China. METHODS: Sixty strains of acinetobacter baumannii were isolated from hospitalized patients between Jul, 2000 and Dec, 2002. Susceptibility of antimicrobial agents and confirmatory tests for Extended-spectrum beta-lactamases (ESBLs) were tested by microdilute method. SHV type beta-lactamases-encoding genes were tested by polymerase chain reaction (PCR). SHV sequences of acinetobacter baumannii HZ02 and HZ10 strains were detected by ABI automated sequencer and were analysed to compare with SHV genes that had been published in GenBank. RESULTS: Eighteen (30.0%) strains of acinetobacter baumannii isolated between Jun, 2001 and Jan, 2002 were carrying SHV beta-lactamases resistant gene of plasmids. Detected SHV sequences of acinetobacter baumannii HZ02 strain and HZ10 strain had 825 and 833 nucleotides respectively and had the same gene sequence as the gene encoding SHV-12 subtype of ESBLs discovered in Switzerland. CONCLUSIONS: Thirty percentage of the clinically isolated acinetobacter baumannii were carrying SHV type (extended-spectrum) beta-lactamases resistant gene of plasmids and causing an outbreak in hospital and was discovered to have carried the strains of SHV-12 subtype producing ESBLs gene in acinetobacter baumannii which was the first reported case in the world.