Endemic Indian clones of Klebsiella pneumoniae-harbouring New Delhi metallo-beta-lactamase-1 on a hybrid plasmid replicon type: A case of changing New Delhi metallo-beta-lactamase plasmid landscapes in India?

Purpose: bla_NDM genes are MBL genes that confer resistance to carbapenems. Globally, they are associated with diverse clones and plasmids. In this study, we characterised three isolates of Klebsiella pneumoniae-harbouring bla_NDM1 from patients undergoing chronic haemodialysis and renal transplantation. Materials and Methods: 3 bla_NDM1-producing K. pneumoniae were isolated from end-stage renal disease patients undergoing haemodialysis and renal transplantation from a nephrology unit. All the three isolates were screened for clinically relevant resistant genes. Plasmid replicon content was analysed by polymerase chain reaction based replicon typing. Conjugation assays were done using azide-resistant Escherichia coli J53 as the recipient strain. Multilocus sequence typing and variable number tandem repeat typing were done to find the clonality. Replicon sequence based typing was attempted to find the diversity of replicon-associated sequences in IncHI3 plasmids. Results: All the 3 bla_NDM positive isolates possessed the New Delhi metallo-beta-lactamase-1 (NDM-1) allele with an IncHI3 plasmid which was not transferable in one isolate. The isolates were found to be sequence type 14 (ST14; 2 nos) and ST38 both of which were previously reported to be the NDM-producing K. pneumoniae STs prevalent in India. Replicon sequence analysis revealed limited sequence diversity within the repHI3 and repFIB locus. Conclusion: To the best of our knowledge, this is the first report of IncHI3, a newly assigned enterobacterial plasmid incompatibility group from India. This could either be a case of importation or a widely circulating NDM plasmid type in India.     

ESCMID-EUCIC clinical guidelines on decolonization of multidrug-resistant Gram-negative bacteria carriers

ScopeThe aim of these guidelines is to provide recommendations for decolonizing regimens targeting multidrug-resistant Gram-negative bacteria (MDR-GNB) carriers in all settings.MethodsThese evidence-based guidelines were produced after a systematic review of published studies on decolonization interventions targeting the following MDR-GNB: third-generation cephalosporin-resistant Enterobacteriaceae (3GCephRE), carbapenem-resistant Enterobacteriaceae (CRE), aminoglycoside-resistant Enterobacteriaceae (AGRE), fluoroquinolone-resistant Enterobacteriaceae (FQRE), extremely drug-resistant Pseudomonas aeruginosa (XDRPA), carbapenem-resistant Acinetobacter baumannii (CRAB), cotrimoxazole-resistant Stenotrophomonas maltophilia (CRSM), colistin-resistant Gram-negative organisms (CoRGNB), and pan-drug-resistant Gram-negative organisms (PDRGNB). The recommendations are grouped by MDR-GNB species. Faecal microbiota transplantation has been discussed separately. Four types of outcomes were evaluated for each target MDR-GNB:(a) microbiological outcomes (carriage and eradication rates) at treatment end and at specific post-treatment time-points; (b) clinical outcomes (attributable and all-cause mortality and infection incidence) at the same time-points and length of hospital stay; (c) epidemiological outcomes (acquisition incidence, transmission and outbreaks); and (d) adverse events of decolonization (including resistance development). The level of evidence for and strength of each recommendation were defined according to the GRADE approach. Consensus of a multidisciplinary expert panel was reached through a nominal-group technique for the final list of recommendations.RecommendationsThe panel does not recommend routine decolonization of 3GCephRE and CRE carriers. Evidence is currently insufficient to provide recommendations for or against any intervention in patients colonized with AGRE, CoRGNB, CRAB, CRSM, FQRE, PDRGNB and XDRPA. On the basis of the limited evidence of increased risk of CRE infections in immunocompromised carriers, the panel suggests designing high-quality prospective clinical studies to assess the risk of CRE infections in immunocompromised patients. These trials should include monitoring of development of resistance to decolonizing agents during treatment using stool cultures and antimicrobial susceptibility results according to the EUCAST clinical breakpoints.     

Variable performance of different commercial systems for testing carbapenem susceptibility of KPC carbapenemase-producing Escherichia coli

ObjectivesThe aim was to evaluate different methods for testing carbapenem susceptibility of Escherichia coli producing KPC-type carbapenemase.MethodsSusceptibility to imipenem, meropenem and ertapenem was assayed using the reference broth microdilution method and several commercial methods (Vitek2, MicroScan, Etest, MIC Test Strip) starting from the same bacterial suspension. Susceptibility to imipenem and meropenem was also tested by Sensititre and disc diffusion (Bio-Rad). Results were interpreted according to EUCAST clinical breakpoints. Essential agreement (EA), category agreement (CA) and error rates were calculated as described by the International Organization for Standardization (ISO) guidelines and also considering the new EUCAST definitions. Genotypic diversity of isolates was evaluated with a RAPD profiling protocol.ResultsOf 54 KPC-positive E. coli isolates, 5.6%, 7.4% and 0% were susceptible standard dosing regimen (S), 55.6%, 72.2% and 0% susceptible increased exposure (I), and 38.9%, 20.4% and 100.0% resistant (R) to imipenem, meropenem and ertapenem, respectively, using the reference broth microdilution method. CA lower than 90% were observed with all systems for imipenem and meropenem using both the ISO and the modified EUCAST criteria. With ertapenem, CA >90% was observed with all methods except Vitek2. RAPD profiling revealed a remarkable genotypic diversity of the isolates, supporting that results were not biased by an oligoclonal nature of the collection.ConclusionsCommercial methods can be unreliable for testing susceptibility to carbapenems of KPC-producing E. coli. Susceptibility should be confirmed by reference broth microdilution.     

Isolation of blaNDM-producing Enterobacteriaceae in a public hospital in Salvador,Bahia, Brazil

Carbapenemases have great importance in the global epidemiological scenario since infections with carbapenemase-producing bacteria are associated with high mortality, especially in hospitalized patients in intensive care units. This study describes two microorganisms producers of the New Delhi Metallo-b-lactamase, Klebsiella pneumoniae and Citrobacter freundii, from two patients admitted to a public hospital in Salvador, Bahia. These are the first clinical cases of New Delhi Metallo-b-lactamase described in microorganisms in the north and northeast Brazil. The isolates were characterized by antimicrobial susceptibility test, with resistance to all β-lactams including carbapenems, negative Modified Hodge Test and the synergy test with Ethylenediaminetetraacetic acid, Phenylboronic Acid and Cloxacillin was positive only with Ethylenediaminetetraacetic acid (difference of >5 mm in the inhibition zone between the disk without and with the inhibitor). Analysis by multiplex PCR for bla_IMP, bla_VIM, bla_NDM, bla_KPC and bla_OXA-48 enzymes confirmed the presence of bla_NDM gene. This report of two different New Delhi Metallo-b-lactamase-producing microorganisms in a different region of Brazil confirms the risk of spreading resistance genes between different species and emphasizes the need for prevention and control of infections caused by these pathogens, which have limited treatment options and have been linked to high mortality rates.     

骨科感染患者病原菌产肺炎克雷伯菌碳青霉烯酶的检测研究

目的对骨科患者感染的大肠埃希菌、阴沟肠杆菌、肺炎克雷伯菌、弗氏柠檬酸杆菌、褪色沙雷菌等进行肺炎克雷伯菌碳青霉烯(KPC)酶检测,为合理使用碳青霉烯类抗菌药物提供依据。方法选择2008年1月-2011年2月骨科感染患者分离的革兰阴性杆菌包括大肠埃希菌110株、阴沟肠杆菌15株、肺炎克雷伯菌145株、弗氏柠檬酸杆菌5株、褪色沙雷菌3株等共278株病原菌采用VITEK-2Compact、K-B纸片法进行药敏试验,以亚胺培南、美罗培南、厄他培南为检测药物,筛选出碳青霉烯类耐药菌株,用改良的Hodge试验筛选、聚合酶链反应(PCR)扩增,确认产KPC酶及基因分型。结果在278株病原菌中,对碳青霉烯类耐药的大肠埃希菌9株、阴沟肠杆菌10株、肺炎克雷伯菌16株,共35株进行改良Hodge试验,肺炎克雷伯菌阳性2株,2株肺炎克雷伯菌PCR扩增出现目的基因片段,其余菌株改良Hodge试验全部阴性。结论该项研究除2株肺炎克雷伯菌中发现目的基因片段,其他病原菌未发现产KPC酶的菌株。     

鲍氏不动杆菌对亚胺培南耐药性及碳青酶烯酶基因型分析

目的了解蚌埠医学院第一附属医院临床分离的鲍氏不动杆菌耐药性及碳青酶烯酶基因型。方法采用琼脂稀释法测定最低抑菌浓度(MICs),用PCR法扩增碳青酶烯酶blaOXA基因和金属酶基因IMP、VIM,对阳性基因型进行全长扩增并测序鉴定。结果临床分离108株鲍氏不动杆菌有56株对亚胺培南耐药,亚胺培南耐药菌株碳青酶烯酶基因型分析显示,检测出blaOXA-23型基因21株,3株检测出blaOXA-58,50株blaOXA-64-like检测阳性,blaOXA-20、blaOXA-24、blaOXA-48、blaOXA-50、blaOXA-55、blaOXA-60检测结果均为阴性,未检测到金属酶IMP、VIM基因。结论临床分离的鲍氏不动杆菌主要产OXA-23、OXA-51型碳青酶烯酶,这可能与临床上经常使用碳青酶烯类抗菌药物有关。     

胶体金免疫层析法快速检测CRE碳青霉烯酶效果评价

目的 评估胶体金免疫层析法(GICA)快速检测耐碳青霉烯类肠杆菌科细菌(CRE)碳青霉烯酶类别的临床应用价值.方法 收集CRE非重复临床分离株73株,采用聚合酶链反应(PCR)鉴定碳青霉烯酶基因型,并采用GICA快速检测CRE的碳青霉烯酶.以PCR为参考方法,评估GICA快速检测CRE碳青霉烯酶的准确性.结果 PCR检...     

耐碳青霉烯类抗菌药物鲍曼不动杆菌膜蛋白机制研究

目的研究耐碳青霉烯类鲍曼不动杆菌膜蛋白机制并了解其流行型别。方法收集仁济医院、瑞金医院和上海市第六人民医院鲍曼不动杆菌共85株,其中亚胺培南和美罗培南耐药菌49株,敏感菌36株。用细菌基因组回文结构重复序列-聚合酶链反应（REP-PCR）分析其流行型别,PCR扩增检测碳青霉烯类水解酶及金属酶,对阳性扩增菌株进行DNA测序分析,同时用超声波和超速离心法提取细菌的膜孔蛋白并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析。结果49株耐亚胺培南和美罗培南鲍曼不动杆菌中,REP-PCR分析结果以A型为主,与敏感菌株存在很大差异;耐药菌株中oxa-51均为阳性,45株为oxa-23阳性;36株敏感菌株中检测到1株oxa-58阳性,29株为oxa-51阳性;金属酶（VIM和IMP-1/4型）均阴性;膜蛋白分析显示,在36株耐药株中38 000附近条带缺失,而在30株敏感株中有17株在相应位置处表达该条带。结论耐碳青霉烯类鲍曼不动杆菌主要以A型流行,产oxa-23水解酶和38 000附近膜孔蛋白条带缺失可能是其主要耐药机制。