Study On The Resistant Mechanism And Nosocomial Infection Control Of Carbapenems Resistant Enterobacteriaceae

Enterobacteriaceae species including E. coli, Klebsiella pneumoniae, Citrobacter freundii and Enterobacter cloacae were the most frequent pathogens of causing nosocomial infections. According to surveillance of bacterial resistance in Shanghai and CHINET, Enterobacteriaceae species accounted for 60% to 70% among gram-negative bacilli. Carbapenems such as imipenem, meropenem and ertapenem showed strong activity against infections caused by multi-drug resistant Enterobacteriaceae, especially extended-spectrumβ-lactamases (ESBLs) and/or AmpC-producing isolates. However, with the widely use of carbapenems in clinic, the occurrence and increase of carbapenem-resistant Enterobacteriaceae (CRE) was unavoidable. The resistant rate of E.coli and K. pneumoniae to carbapenem increased to 1.0%, and in some Enterobacter spp. the resistant rate has already reached 7.0%. The resistant rate of some nonfermentative Bacilli was also about in 20%-30%. The carbapenems met an urgent challenge in clinic because most of carbapenems-resistant bacteria were also resistant to other antimicrobial agents.Among all the CRE isolates from 2005 to 2009 in Huashan Hospital, we often isolated CRE strain from different patients in the same bed in a ward. In Aug.2005, Jan 2006, Mar 2007, and Sep 2007,3 C. freundii isolates and 1 K. pneumoniae strain were collected from four different patients in a bed in a neurosurgery ward. Different species of CRE isolates were also isolated from the same one patient, including Acinetobacter baumannii and K. pneumoniae, C. freundii and K. pneumoniae, K. pneumoniae and E. coli. Therefore, we presumed there were two major mechanisms underlying the widespread dissemination of CRE isolates in hospital. One was horizontal transmission, plasmid harboring carbapenemase gene, such as KPC-2 type carbapenemase, was transmitted between different species directly. The other was clone transmission, CRE isolates were widely disseminated in different patients through various ways and caused outbreak. Currently, most of studies have focused on resistant mechanism and only few are associated with the mechanism for the widespread dissemination of CRE isolates between different species and nosocomial infection despite the frequency of CRE isolates is increasing year by year. A survey by Kochar revealed that improved decontamination of hands and environmental surfaces could reduce the spread of carbapenem-resistant K. pneumoniae successfully. However, such study has been not reported in our country. Infections caused by CRE isolates, especially bacterial meningitis, have usually been very serious and antibiotic choices may be quite limited. Compared with previous years, the frequency of carbapenems-resistant K. pneumoniae increased dramatically in 2009. We presumed the outbreak of CRE isolates would be unavoidable in hospital environment if the effective actions of infection control were still lack. Therefore, it was urgent to study the mechanism of CRE dissemination between different species and take effective infection control actions immediately to limit the occurrence and outbreak. In order to provide a basis for controlling outbreak and spread of CRE isolates, our study aims to reveal the mechanism of CRE dissemination between different species through molecular and epidemiological study. These may have a positive impact on improving clinical therapy, rational drug use and rehabilitation of patients. This study included four parts as follows.Carbapenemase-producing was the main resistant mechanism of CRE isolates to carbapenems. Some studies also reaveled that extended-spectrum P-lactamases (ESBLs) and/or AmpC-producing coupled with outer membrane porin loss in CRE isolates is another main resistant mechanism to carbapenems. In 2009, CLSI recommends modified Hodge testing (MHT), which initially used to detect penicillinase-positive Neisseria gonorrhoeae by Wavell Hodge, for detecting carbapenemase-producing isolates among Enterobacteriaceae. Then, MHT was used to detect metallo-β-lactamase in Pseudomonas aeruginosa and A. baumannii and CMY-1 type AmpC in K. pneumoniae and E. coli.In 2005, the first CRE strain was isolated in Huashan Hospital. Up to Dec.2009, 220 CRE isolates were collected by the surveillance of bacterial resistance, including K. pneumoniae (158), K. oxytoca (3), C.freundii (35), Citrobacter spp. (6), E. coli (6), Enterobacter cloacae (4), Proteus mirabilis (3), Serratia marcescens (2), Enterobacter aerogenes (1), Bordetella bronchiseptica (1) and Providencia stuartii (1). The result of antimicrobial susceptibility testing indicated that the frequency of K. pneumoniae isolates resistant to imipenem, meropenem and ertapenem was all below 2%, but in 2009 quickly increased to 16%. The frequency of C. freundii resistant to carbapenems was about 10% in 2005, but increased to 45% in 2006, and 35% during 2007 to 2009. The antimicrobial susceptibility testing of 78 CRE isolates from Jan. 2005 to Apr.2009 by agar dilution indicated that the susceptibility rate of imipenem, meropenem, ertapenem and colistin was 16.4%,17.9%,1.3% and 96.0%, respectively. The MIC50/MIC90 of imipenem, meropenem, ertapenem and colistin against 78 CRE isolates was 32/256 mg/L,64/>256 mg/L,128/>256 mg/L and 1/1 mg/L. The result of MHT indicated that 93.6%(73/78) of these CRE isolates were carbapenemase-producing.The result of detection of carbapenemases, ESBLs and plasmid-borne AmpC gene by PCR method and DNA sequencing indicated that 33.3%(26/78) were KPC-2 type carbapenemase-producing isolates, in which 7.7%(2/26),7.7%(2/26),3.8% (1/26) were coupled with VIM-1, IMP-2, IMP-1 metallo-β-lactamase, respectively. In addition,6.4%(5/78),5.1%(4/78),3.8%(3/78),6.4%(5/78),12.8%(10/78) were VIM-1, IMP-1, IMP-2, GIM, OXA-69 type carbapenemase-producing isolates, respectively. The result of multiple PCR analysis indicated,20.5%(16/78) were OXA-23-like, OXA-51-like or OXA-58-like carbapenemase-producing isolates. VIM-2, SPM, NMC, IMI, IND, OXA-48, OXA-50, OXA-55, OXA-60 and OXA-24-like type carbapenemases genes were all negative.According to Tenover criteria,25 C. freundii isolates belonged to 4 different DNA patterns and in which 64.0%(16/25) isolates were the same genotype. The 43 strains of K. pneumoniae belonged to 10 different DNA patterns,6 KPC-2 type carbapenemase-producing isolates were the same genotype and other 3 isolates were different. Interestingly, some KPC-2 type carbapenemase negative isolates were also clonally related.Compared with carbapenem-susceptible Enterobacteriaceae,29.5%(23/78) CRE isolates presented one single band, probably corresponding to OmpK35 or OmpK36 porin, suggesting that they have lost at least one porin.60.3%(47/78) CRE isolates were down-regulation of the OmpK35 or OmpK36 porin.The result of conjugation experiment with 6 strains of KPC-2 type carbapenemase-producing K. pneumoniae (including 2 strains of KPC-2 type carbapenemase-producing coupled with VIM-1 type metallo-β-lactamase-producing K. pneumoniae) and 3 strains of carbapenem-resistant C. freundii indicated that transconjugants of all 9 isolates were obtained from selective plate. The result of antimicrobial susceptibility testing andβ-lactamase gene detection by PCR method indicated ESBL or AmpC gene were successfully transferred from donor to recipient by conjugation. Transconjugants were all resistant to third cephalosporins. However, KPC-2 type carbapenemase gene and VIM-1 type metallo-β-lactamase gene were both not transferred. The transformation testing of KPC-2 type carbapenemase gene and VIM-1 type metallo-β-lactamase gene also failed. We presumed that the carbapenemase gene may locate on chromosome or the plasmid bearing KPC-2 type carbapenemase gene was too large to be transferred.The same CRE strain was often isolated from different patients in the same bed, and different species of CRE isolates were also isolated from the same one patient. We also found some K. pneumoniae strains developed carbapenem resistance during therapy. We tried to reproduce in vitro the phenomenon observed in vivo and hoped to explain the mechanism of wide CRE dissemination between different species. The transfer experiment of carbapenemase gene was performed by conjugation. The donor and recipient were pan-drug resistant A. baumannii, and carbapenems-susceptible K. pneumoniae, respectively. Transconjugants were obtained after selection on ertapenem and sulbactam, respectively. The result of antimicrobial susceptibility testing indicated that transconjugants were resistant to imipenem, meropenem and ertapenem. ERIC-PCR and PFGE demonstrated that transconjugants and recipient were the same genotype. Because the type of metalloenzyme gene was unknown in donor, the resistant mechanism of transconjugants to carbapenems still needs further research. We should select more pan-drug resistant isolates for reproduction in vitro the phenomenon observed in vivo. The resistant plasmids of transconjugants, donor and recipient should be sequenced in order to reveal the transfer mechanism of carbapenemase.An epidemiological survey was conducted for different patients who infected or colonizated by CRE isolate in the same one bed in a neurosurgery ward. The specimen were cultured in 5 mL MH broth containing one piece of ertapenem (10μg). We isolated the CRE from rectal swab sample, meatus urinarius and the water in oxygen filter system. PFGE demonstrated that three CRE isolates were the same genotype. After that, we isolated 5 carbapenems-resistant isolates, including 2 K. pneumoniae isolates,3 A. baumannii isolates and 1 S. maltophilia isolate from the water in five oxygen filter system in this ward. The results of screening CRE isolates in specimens from hospital environment and medical staff in neurosurgery ward indicated that the handrails of bed, the hands of nurse and the screen of infusion pump were all positive for carbapenems-resistant K. pneumoniae. Therefore, we presumed that the CRE isolates in oxygen filter system and the hospital environment may the key source of various infections, especially respiratory tract infections.The retrospective review of the medical history of patients with CRE infection or colonization indicated that prior antimicrobial agents use, presence of a variety of catheters may contribute to CRE infection and difficult to eradicate. One of the key factors for the spread of CRE isolates in hospital was the frequent exchange of patients with CRE infection or colonization between different beds or wards. Conclusions:1. The result of antimicrobial susceptibility testing indicated that most of CRE isolates including K. pneumoniae and C.freundii in Huashan Hospital were highly resistant to imipenem, meropenem and ertapenem. The MIC90 of imipenem, meropenem and ertapenem was all above 256 mg/L. Most of CRE isolates were isolated from patients in neurosurgery. The main source of specimens was urine and respiratory tract.2. The results of detection of carbapenemase in 78 CRE isolates by MHT indicated, 93.6%(73/78) were carbapenemase-producing isolates. Compared with PCR method, the sensitivity and specificity of detection of carbapenemase by MHT were 97.9% and 12.9, respectively.3. The results of resistance gene detection and outer membrane porin analyzed by SDS-PAGE indicated,60.3%(47/78) were carbapenemases-producing isolates in which 33.3%(26/78) isolates producing KPC-2 type carbapenemase. Production of carbapenemases is the main mechanism for carbapenem resistance in Enterobacteriaceae isolates; 35.9%(28/78) isolates have lost at least one porins. Production of ESBL and/or plasmid-borne AmpCβ-lactamases coupled with porin loss is another important resistant mechanism.4. Although several conjugation and transformation experiments were performed, KPC-2 type carbapenemase still did not transfer from donor to recipient except for ESBLs. We presumed that the KPC-2 type carbapenemase may locate on chromosome or the plasmid bearing KPC-2 type carbapenemase gene is too large to be transferred from donor to recipient.5. The results of PFGE suggest clone spread of CRE isolates between different wards, including both C.freundii and K. pneumonia.6. Compared with direct method,5 mL of MH broth containing 10μg of ertapenem can be used in epidemiologic study because it may increase the detection rate of CRE isolates in all kinds of specimens.7. Many oxygen filter systems harbor carbapenems-resistant isolates, including K. pneumoniae, A. baumannii and S. maltophilia. The CRE isolates were also isolated in environmental surfaces of bed, nursing staff's hands and the rectal in patient. All of above factors may the key source of resulting in various infections, especially respiratory tract infections.8. Our research indicated that improved hand hygiene and environmental surface cleaning may be the key procedure to limit the spread of carbapenems-resistant isolates. It is urgent to take effective procedures to control infections due to carbapenem-resistant or carbapenemase-producing Enterobacteriaceae in hospital environment.