Prevalence And Transfer Mechanism Of 16S RRNA Methylase Genes Among Escherichia Coli And Klebiella Pneumonia Clinical Isolates

BackgroundEscherichia coli and Klebiella pneumonia are the most common pathogens which cause hospital-acquired infections, such as respiratory tract infection, urinary tract infection, septicaemia, pyogenic infection and so on. The infections caused by E. coli and K. pneumonia deserve clinical concern. With the increase of antimicrobials used for treating the infections caused by bacteria, the multi-resistance of E.coli and K. pneumonia to antimicrobials has been increasing, which results in the difficulty in treating the infections by bacteria and controlling the dissemination of infections. In particular, E. coli and K. pneumonia can produce extended-spectrum beta lactamases (ESBLs) which can hydrolyze penicillins, cephalosporins and aztreonam. In addition to ESBLs, E. coli and K. pneumonia also can produce AmpCs. Recently, it has been reported that E.coli and K. pneumonia can produce carbapenemases including KPCs and metallo enzymes which can hydrolyze all?-lactam agents including carbapenems. As aminoglycosides can cause ototoxicity and nephrotoxicity, aminoglycosides are limited for treating the infections. However, aminoglycosides have long post antibiotics effect to E. coli, K. pneumonia and Pseudomonas aeruginosa and usually are used for treating infections caused by multi-resistant bacteria in combination with?-lactam agents or fluoquinolones. Antimicrobial activity of aminoglycosides depends on binding to a highly conserved motif of 16S rRNA. The mechanism of resistance to aminoglycosides is frequently due to the acquisition of modifying enzymes that vary in their substrate ranges, such as acetyltransferases, phosphorylases and adenylyltransferases. Recently, production of 16S rRNA methylases has emerged as a novel mechanism of high-level resistance to most of clinically important aminoglycosides, including arbekacin, amikacin, tobramycin and gentamicin. Since the first 16S rRNA methylase, ArmA, was found in 2003, six plasmid-mediated 16S rRNA methylases (ArmA, RmtA, RmtB, RmtC, RmtD, and NpmA) have been found in Gram-negative bacilli clinical isolates. Recently, the seventh 16S rRNA methylase, RmtE, was found in bovine-origin E. coli isolates. The infections caused by Gram-negative bacilli clinical isolates producing 16S rRNA methylases were reported in many countries worldwide. The prevalence of 16S rRNA methylases among Gram-negative bacilli clinical isolates in China, especially among Enterobacteriaceae, is not known. The aim of this study was to investigate the occurrence and transfer mechanism of 16S rRNA methylase genes in E. coli and K. pneumonia clinical isolates from a teaching hospital in Wenzhou, China.Part I:Prevalence and characteristic of 16S rRNA methylase genes among E. coli and K. pneumonia clinical isolatesThree hundred and thirty-seven non-duplicate K. pneumoniae isolates isolated from January 2006 to September 2007 and 680 non-duplicate E. coli clinical isolates isolated from January 2006 to July 2008 were consecutively collected from the inpatients in a teaching hospital in Wenzhou, China. The clinical isolates were identified by using Gram stain, GNI cards on the VITEK-60 system and biochemical assays. Sixty-four K. pneumoniae clinical isolates and 365 E. coli clinical isolates with resistance to at least one of gentamicin, amikacin, and tobramycin based on the criteria following CLSI, were first screened for potential 16S rRNA methylase producers. Of 337 K. pneumoniae isolates,64(19.0%),28(8.3%), and 55(16.3%) isolates were resistant to gentamicin, amikacin and tobramycin, respectively,357 (52.5%),346 (50.9%) and 44(6.5%)isolates among 680 E.coli isolates, respectively. All amikacin-resistant isolates were concomitantly resistant to tobramycin and gentamicin. Twenty-one K. pneumoniae isolates and 37 E. coli isolates were positive for 16S rRNA methylase genes determined by PCR and DNA sequencing, among which 3(0.9%,3/337),13(3.9%,13/337) and 5(1.5%,5/337) K. pneumoniae were for armA, rmtB and both armA and rmtB, respectively, and 36 and one E. coli isolate for rmtB and armA. The positive rates of 16S rRNA methylase genes among overall, amikacin-, tobramycin- and gentamicin-resistant K. pneumoniae isolates were 6.2% (21/337),75.0%(21/28),38.2%(21/55) and 32.8%(21/64), respectively,5.4% (37/680),84.1%(37/44),10.4%(37/357) and 10.7%(37/364) among E. coli isolates, respectively. The rmtA, rmtC, rmtD and npmA genes were not detected in any of tested isolates. All 16S rRNA methylase gene-positive isolates were highly resistant to gentamicin, amikacin and tobramycin (MICs,?256?g/mL). Nineteen K. pneumoniae isolates harboring 16S rRNA methylase genes were ESBL producers. Twenty-one,15, and 19 ESBLs-producing K. pneumoniae isolates were positive for blaTEM, blaSHV, and blaCTX-M, respectively. All blaTEM amplicons were blaTEM-1. Nine, 2,1, and 3 isolates harbored blaSHV-12, blaSHV-1, blaSHV-2 and blaSHV-11, respectively. All 19 ESBLs-producing isolates were found to harbor blaCTX-M, including 13 isolates harboring blaCTX-M-14-and 6 isolates harboring bla CTX-M-15. Thirty-one and 37 E. coli isolates were found to harbor blaCTX-M and blaTEM.All blaTEM type genes were narrow spectrum?-lactamase gene, blaTEM-1. Twenty-four,11, three, one and one isolates were positive for blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTx-M-3 and blaCTX-M-24, respectively. Among which seven isolates harbored both blaCTX-M-14 and blaCTX-M-15 and two isolates harbored both blaCTX-M-27 and blaCTX-M-14. All 16S rRNA methylase gene-positive isolates were positive for class I integonase gene16S rRNA methylase genes, armA and rmtB, were commonly identified among K. pneumoniae and E. coli clinical isolates in a Chinese teaching hospital. Coexistence of two different 16S rRNA methylase genes in same isolate was first found among clinical isolate. armA and rmtB usually co-exist with ESBL genes and class I integonase gene in same isolate.Part?:The transfer mechanism of 16S rRNA methylase genesHigh-level aminoglycoside resistance could be transferred by conjugation from 2 of 3 armA-,8 of 13 rmtB- and 4 of 5 both armA and rmtB -positive K. pneumionae donors. The aminoglycoside-resistant plasmids harboring rmtB of 4 E.coli isolates could be transferred into recipients by conjugation by repeat attempts. All transconjugants and transconjugants were highly resistant to amikacin, gentamicin and tobramycin(MICs,?256?g/mL) and positive for class I integonase gene. The genotypes of?lactamase genes of transconjugants and transconjugants were same that their donors. Only the plasmids carrying rmtB were obtained by conjugation and transformation from the K. pneumoniae isolates carrying both armA and rmtB.Four K. pueumoniae isolates harboring both armA and rmtB contained only one 75-kb transferable plasmid whereas the other isolate contained three plasmids with a range of sizes other than a 75-kb plasmid. The 75- kb plasmids and chromosomal fragments of 5 donors harboring both armA and rmtB and 4 respective transconjugants hybridized with rmtB-specific probe. The hybridization signals for armA-specific probe were only obtained on the chromosomal fragments of the 5 K. pneumoniae donors. The 54-kb plasmids of the armA-positive E. coli donor and its transformant hybridized with the armA-specific probe. The hybridization signals for rmtB-specific probe were obtained on the 54-kb plasmids of the E. coli isolates harboring rmtB and its t transformant. The upstream of the armA gene in K. pneumoniae and E. coli clinical isolates contained ISCR1 and putative transposase gene, tnpU. Another putative transposase gene, tnpD was located downstream of the armA. The structural gene for the rmtB gene in K. pneumoniae clinical isolate was preceded by a transposon, Tn3, including tnpA encoding transposase, tnpR encoding resolvase, and blaTEM-1 encoding beta-lactamase. A transposase gene, tnpA,fib gene involved in fertility inhibition of IncP plasmid proteins, and tral gene encoding conjugal nickase and helicase involved in conjugation were located downstream of the rmtB gene. The rmtB gene was flanked by two transposase gene, tnpA comprising insertion sequence (IS26). The upstream of the rmtB gene in E.coli clinical isolate was similar to that in K. pneumoniae clinical isolate. However, the downstream of rmtB gene in E. coli clinical isolate was different from that in K. pneumoniae. A transposase gene, tnpA, and a quinolone efflux pump gene, qepA, located the downstream of the rmtB gene in E. coli clinical isolate.armA and rmtB in K. pneumoniae clinical isolates usually co-transfer with ESBL genes and class?integonase gene on self-transmissible conjugative plasmids to recipients. However, armA and rmtB in E. coli clinical isolates usually co-exist with ESBL genes and class?integonase gene on non-transmissible plasmids. The rmtB genes were located on the plasmids and the armA genes were probably located on the chromosomes in 5 both armA and rmtB-positive isolates. rmtB and armA located between the insert sequences. The self-transmissibility of plasmids in K. pneumoniae clinical isolates was associated with tral located on the downstream of rmtB. The resistance of E. coli clinical isolates with rmtB to fluoquinolones was associated with qepA located on the downstream of rmtB.Part?Homology analysis on K. pneumoniae and E. coli clinical isolates harboring 16S rRNA methylase genesThe homology of K. pneumoniae and E. coli clinical isolates harboring 16S rRNA methylase genes was determined by PFGE. The 21 K. pneumoniae isolates were grouped to 14 clonal patterns by PFGE, designated patterns A to N. The predominant patterns were type A and type?. Among 13 rmtB-positive isolates,5 rmtB-positive isolates (4 from ICU and one from neurosurgery ward) belonged to type A and the remaining 8 isolates belonged to 8 different patterns. Of 5 both armA and rwtB-positive isolates,4 isolates (3 from chest surgery unit and one from urology unit) belonged to type?and the remaining isolate from neurology unit belonged to type B. Other patterns were represented by a single isolate from separate clinical wards. The 37 E. coli isolates carrying 16S rRNA methylase genes were grouped to 19 clonal patterns by PFGE, designated PFGE type A to S. The predominant PFGE type was H (12 isolates). The 12 PFGE type H isolates distributed seven different wards, while eight isolates isolated from ward 11 belonged to seven separate PFGE types. Thirty-seven 16S rRNA methylase gene-positive isolates distributed 19 different clinical units with separate PFGE typesIn addition to a small clonal dissemination (12 PFGE type H isolates), most of 16S rRNA methylase gene-positive isolates distributed distantly related patterns and separate clinical units, which showed considerable molecular heterogeneity. Both horizontal gene transfer and clonal spread were responsible for the dissemination of rmtB and armA in China.ConclusionAmikacin has high antibacterial activity in vitro and is indicative of 16S rRNA methylases; High prevalence of 16S rRNA methylase genes, especially rmtB, was found among K. pneumoniae and E. coli clinical isolates from Wenzhou, China; Coexistence of two different 16S rRNA methylase genes is first found in the same isolate; armA and rmtB in K. pneumoniae clinical isolates usually co-transfer with ESBL genes and class?integonase gene on self-transmissible conjugative plasmids to recipients. However, armA and rmtB in E. coli clinical isolates usually co-exist with ESBL genes and class?integonase genon non-transmissible plasmids; rmtB and armA locate between the insert sequences; The upstream of the rmtB gene in E. coli clinical isolate was similar to that in K. pneumoniae clinical isolate. However, the downstream of rmtB gene in E. coli clinical isolate was different from that in K. pneumoniae; Both horizontal gene transfer and clonal spread were responsible for the dissemination of rmtB and armA in China.