In vitro activity of rifaximin against clinical isolates of Escherichia coli and other enteropathogenic bacteria isolated from travellers returning to the UK

Rifaximin is licensed in the EU and USA for treating travellers’ diarrhoea caused by non-invasive bacteria. Selection for resistance mechanisms of public health significance might occur if these are linked to rifamycin resistance. Rifaximin MICs were determined by agar dilution for 90 isolates each of Escherichia coli, Shigella spp., nontyphoidal Salmonella enterica, typhoidal S. enterica and Campylobacter spp., an additional 60 E. coli with CTX-M ESBLs isolated from patients with travellers’ diarrhoea, and 30 non-diarrhoeal carbapenemase-producing E. coli. Comparators were rifampicin, ciprofloxacin, azithromycin, trimethoprim/sulfamethoxazole and doxycycline. Isolates with rifaximin MICs>32 mg/L were screened for arr genes, and critical rpoB regions were sequenced. Rifaximin was active at ≤32 mg/L against 436/450 (96.9%) diverse Enterobacteriaceae, whereas 81/90 (90%) Campylobacter spp. were resistant to rifaximin at ≥128 mg/L. Rifaximin MICs were ≥128 mg/L for two Shigella and five MDR E. coli producing NDM (n = 3), OXA-48 (n = 1) or CTX-M-15 (n = 1). Two of the five MDR E. coli had plasmids harbouring arr-2 together with bla_NDM, and two (one each with bla_NDM and bla_CTX-M-15) had His526Asn substitutions in RpoB. The rifamycin resistance mechanism remained undefined in one MDR E. coli isolate (with bla_OXA-48) and the two Shigella isolates. Rifaximin showed good in vitro activity against diverse Enterobacteriaceae but was largely inactive against Campylobacter spp. Rifaximin has potential to co-select MDR E. coli in the gut flora, but much stronger associations were seen between ESBL and/or carbapenemase production and resistance to alternative treatments for travellers’ diarrhoea, notably ciprofloxacin and azithromycin.     

Worldwide dissemination of acquired carbapenem-hydrolysing class D β-lactamases in Acinetobacter spp. other than Acinetobacter baumannii

The aim of this study was to identify acquired OXA-type carbapenemases in Acinetobacter spp. other than Acinetobacter baumannii. From a total of 453 carbapenem-susceptible and -resistant Acinetobacter isolates collected worldwide, 23 were positive for bla_OXA genes by multiplex PCR. These isolates were identified as Acinetobacter pittii (n = 18), Acinetobacter nosocomialis (n = 2), Acinetobacter junii (n = 1) and Acinetobacter genomic species 14TU/13BJ (n = 2). The bla_OXA genes and associated insertion sequence (IS) elements were sequenced by primer walking. In 11 of these isolates, sequencing of the PCR products revealed that they were false-positive for bla_OXA. The remaining 12 isolates, originating from Europe, Asia, South America, North America and South Africa, harboured OXA-23 (n = 4), OXA-58 (n = 5), OXA-40-like (n = 1) and OXA-143-like (n = 1); one A. pittii isolate harboured both OXA-23 and OXA-58. IS elements were associated with bla_OXA in 10 isolates. OXA multiplex PCR showed a high degree of false-positive results (47.8%), indicating that detection of bla_OXA in non-baumannii Acinetobacter spp. should be confirmed using additional methods.     

Genotypes,carbapenemase carriage,integron diversity and oprD alterations among carbapenem-resistant Pseudomonas aeruginosa from Russia

Pseudomonas aeruginosa is a serious opportunistic pathogen demonstrating a high level of resistance to many groups of antibiotics, including carbapenems. This study aimed to characterise the molecular epidemiology and prevalence of mobile genetic elements associated with resistance to carbapenems among P. aeruginosa (CRPA) clinical isolates. Among 145 carbapenem-resistant P. aeruginosa isolates, 34 different sequence types (STs) were detected; the six most common STs were ST654 (24%), ST235 (24%), ST111 (8%), ST446 (6%), ST357 (5%) and ST2592 (a novel single-locus variant of ST357) (4%). A carbapenemase gene was found in 94 isolates (64.8%). The bla_VIM-2 gene was harboured by 64 isolates (44.1%) restricted to ST111, ST235 and ST654, and the bla_GES-type and bla_OXA-10 group genes were each detected in 15 isolates (10.3%); none of other tested carbapenemase genes, including bla_IMP, bla_NDM and bla_GIM, were detected. Among the bla_VIM-2-positive isolates, five types of bla_VIM-2-containing integrons were discovered, including In56, In559, In59-like, In59 and In249. The oprD gene was disrupted by an insertion sequence (IS) in 15.9% of isolates. Overall, five types of IS elements were found (ISPsme1, ISPa1328, ISPa26, ISPst2 and ISPa195). Observed rearrangements within variable regions of bla_VIM-2-carrying integrons in conjunction with the discovery of a novel type of oprD-disrupting IS element illustrate the ongoing evolution of CRPA a, which warrants further investigation.     

Multiclonal spread of VIM-1-producing Enterobacter cloacae isolates associated with In624 and In488 integrons located in an IncHI2 plasmid

Over a 6-year period (2007–2012), the emergence of Enterobacter cloacae isolates resistant to β-lactams and with reduced susceptibility to carbapenems was observed in Hospital Universitario 12 de Octubre (Madrid, Spain). To determine the possible role of metallo-β-lactamases (MBLs) in the resistance profile of these isolates, a molecular and clinical epidemiological study was performed, including determination of patients’ clinical characteristics, genetic diversity of strains, resistance mechanisms to carbapenems, and the genetic environment of VIM-1. A total of 73 E. cloacae isolates showed resistance to extended-spectrum cephalosporins and reduced susceptibility to at least one carbapenem during 2007–2012. PCR amplification revealed the presence of bla_VIM-1 gene in 37 isolates, bla_VIM-2 in 1 isolate and bla_KPC in 5 isolates. Molecular typing showed high clonal diversity of E. cloacae isolates carrying bla_VIM-1. The genetic environment of bla_VIM-1 was investigated and two integron structures were found: intI–bla_VIM-1–aacA4–dfrB1–aadA1–catB2–qacEΔ1/sul1 (In624); and intI–bla_VIM-1–aacA4–aadA1–qacEΔ1/sul1 (In488). Isolates belonging to three clones (A, F and G) harboured different types of integron (In624 or In488) despite belonging to the same clone. Conjugal experiments showed an association with a conjugative plasmid of ca. 300 kb belonging to IncHI2 group, which is common in Spanish hospitals, suggesting that the widespread dissemination of bla_VIM-1 may be due to horizontal transfer of mobile genetic determinants rather than the result of spreading of a few clones. These results have implications for infection control programmes in the hospital.     

Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-β-lactamase-1-producing Klebsiella pneumoniae in the neonatal intensive care unit of an Indian hospital

Emergence of multi-drug resistance, especially carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to public health. The aim of this study was to characterize CRKP isolates from infants admitted to the Neonatal Intensive Care Unit (NICU) to find the clonal outbreak of New Delhi metallo-β-lactamase (NDM) producers. In this study 17 CRKP isolates were analysed. Antimicrobial susceptibility of the isolates was determined by the disc diffusion and micro-dilution method. Carba-NP test and double-disk synergy test (DDST) were performed for the detection of carbapenemase and metallo-β-lactamase-producing K. pneumoniae. Antibiotic-resistant markers were detected by polymerase chain reaction (PCR) followed by sequencing. Clonal relatedness of the isolates was checked by multi-locus sequence typing. Conjugation experiments were performed to determine the transferability of the plasmids. All 17 CKRP isolates were found to carry bla_NDM (13 bla_NDM-1, 1 bla_NDM-4 and 3 bla_NDM-5), seven isolates carried bla_OXA-48, 13 isolates had bla_CTX-M-15, seven isolates carried bla_CMY-1 and five isolates were found to carry bla_SHV-1 on conjugative plasmids of different types (IncFIA, IncFIB, IncFIIAs, IncFIC, IncA/C, IncF, IncK, IncX, IncW and IncY). Of six different sequence types (STs) identified, ST3344 was detected as a novel ST in two K. pneumoniae isolates. Genetic environment analysis revealed ISAba125 and bleomycin genes flanking to all bla_NDM variants. This is the first report of novel ST3344 in two NDM-1-producing K. pneumoniae isolates from neonates admitted to the NICU of a North Indian Hospital. This study is provides understanding of the genetic features of this newly emerged strain type.     

Salmonella enterica isolated from infections in Australian livestock remain susceptible to critical antimicrobials

Salmonella enterica is a zoonotic pathogen causing a variety of diseases in humans and animals. Many countries are reporting an increase in the prevalence of multidrug-resistant (MDR) S. enterica in food animals. The aim of this study was to determine whether S. enterica isolated from livestock in New South Wales, Australia, have similar resistance traits to those reported internationally. Salmonella enterica (n = 165) from clinical infections in food animals between 2007 and 2011 were serotyped and tested for susceptibility to 18 antimicrobials. Also, 22 antimicrobial resistance genes (ARGs), 3 integrons and 18 plasmid replicon types were screened for using PCR. Most isolates (66.1%) remained susceptible to all antimicrobials; 8.5% of the isolates were resistant to four or more antimicrobials. Antimicrobials with the highest prevalence of resistance were sulfafurazole (28.5%), ampicillin (17.0%), tetracycline (15.8%) and trimethoprim (8.5%). There was no resistance to fluoroquinolones or third-generation cephalosporins. The most common ARGs were bla_TEM (15.2%), sul2 (10.3%), tetB (9.1%), tetA (5.5%), aphA1 (4.8%) and dhfrV (4.8%). Class 1 integrons (7.9%) and IncFIIA (69.7%) were the most commonly detected integron and plasmid replicon types, respectively. Class 1 integrons were positively associated with MDR phenotypes and ARG carriage (P ≤ 0.001). Internationally prominent MDR serovars associated with severe disease in humans (e.g. AmpC-positive Salmonella Newport) were not detected. Overall, the comparatively favourable resistance status of S. enterica in Australian livestock represents minimal public health risk associated with MDR strains and supports a conservative approach to the registration of antimicrobial drug classes in food-producing animals.     

Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Chinese hospitals

We investigated the molecular epidemiology and carbapenem resistance mechanisms of 258 non-duplicate carbapenem-resistant clinical isolates of Pseudomonas aeruginosa collected from 2006 to 2007 at 28 hospitals in China. Up to 88% of the carbapenem-resistant isolates were multidrug-resistant. Pulsed-field gel electrophoresis (PFGE) revealed that levels of intrahospital and interhospital dissemination of clones were low. To assess the mechanisms leading to resistance, all 258 carbapenem-resistant isolates were analysed for expression of the chromosomal β-lactamase (AmpC), the porin important for entry of carbapenems (OprD) and an efflux system (MexAB-OprM) known to extrude some β-lactams. Carbapenem resistance was driven mainly by mutational inactivation of OprD, accompanied or not by hyperexpression of AmpC or MexAB-OprM. Metallo-β-lactamase genes were detected in 22 carbapenem-resistant isolates in China, belonging to eight pulsotypes. The bla_OXA-50 gene was detected among all of the carbapenem-resistant isolates, whereas the bla_GES-5 gene was detected in only one carbapenem-resistant isolate.     

Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae

A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n = 17); Klebsiellapneumoniae (n = 3); Enterobacter spp. (n = 6); Acinetobacter genospecies 3 (n = 1); Acinetobacterbaumannii (n = 1); Pseudomonasaeruginosa (n = 2); and Stenotrophomonasmaltophilia (n = 2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla_SHV-5 in a bla_OXA-23-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.     

Carbapenemase-producing Enterobacteriaceae in a tertiary hospital in Madrid,Spain: high percentage of colistin resistance among VIM-1-producing Klebsiella pneumoniae ST11 isolates

Here we describe the carbapenemase genes, genetic relatedness and antimicrobial susceptibility data of 123 carbapenemase-producing Enterobacteriaceae (CPE) clinical isolates recovered from 2010 to 2012, comprising Klebsiella pneumoniae (n = 79), Klebsiella oxytoca (n = 13), Serratia marcescens (n = 14), Enterobacter cloacae (n = 12), Enterobacter asburiae (n = 4) and Enterobacter aerogenes (n = 1). VIM-1 was the most common carbapenemase (n = 101) followed by KPC-2 (n = 19), OXA-48 (n = 2) and IMP-22 (n = 1). Among the K. pneumoniae isolates, nine sequence types (STs) were identified but two clones were dominant: ST11 (54/79) containing mainly VIM-1-producing isolates; and ST101 (13/79) constituted by KPC-2-producing strains. Pulsed-field gel electrophoresis (PFGE) showed a higher genetic diversity among the remaining Enterobacteriaceae. Amikacin and fosfomycin were the most active agents with 82.9% and 80.5% susceptibility, respectively. Non-susceptibility to tigecycline was detected in 36.5% of strains. Overall, colistin resistance was 24.7% and was as high as 47% in Enterobacter spp. An increase in colistin resistance from 13.5% to 31.7% was observed among K. pneumoniae isolates during the study period. Resistance was focused on ST11 since 83.3% of colistin-resistant strains belonged to this clone. The high level of colistin resistance observed in this study is worrying with respect to the already limited therapeutic options for infections caused by multidrug-resistant Gram-negative bacteria.     

碳青酶烯类耐药肺炎克雷伯菌耐药基因检测及同源性分析

目的 探讨浙江省台州医院分离的碳青霉烯类耐药肺炎克雷伯菌（carbapenem-resistant Klebsiella pneumonia,CRKP）的耐药基因分型,并进行同源性分析。方法 收集浙江省台州医院2017年1月-2017年11月临床分离非重复的41株CRKP,用VITEK-compact2全自动微生物分析仪进行鉴定及药敏试验,质谱仪VITEK MS和纸片扩散法（K-B法）分别对鉴定、药敏进行复核;采用表型筛选试验对试验菌进行产A、B类碳青霉烯酶筛选;PCR检测耐药基因型;目的产物经基因测序和BLAST网上比对确定其基因型;脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分析41株菌株同源性。结果 表型筛选试验提示41株CRKP均产碳青霉烯酶;检出bla_KPC、bla_IMP、bla_NDM基因阳性率分别为95.12%（39/41）,4.88%（2/41）,2.44%（1/41）,未检测到bla_SIM、bla_SPM、bla_VIM、bla_GIM基因;测序结果bla_KPC、bla_NDM、bla_IMP基因型别为bla_KPC-2、bla_NDM-1、bla_IMP-4;PFGE结果可分为A~N共14个谱型,以A型41.46%（17/41）、B型21.95%（9/41）、C型9.76%（4/41）为主,A型主要分布于神经外科,B型、C型主要分布于重症医学科。结论 浙江省台州医院CRKP耐药基因型以bla_KPC-2为主,且存在克隆株传播。医院感染控制部门及临床各科室应引起重视,采取有效措施,控制耐药株的传播。     

Spread of imipenem-resistant Acinetobacter baumannii of European clone II in Western China

The aim of this study was to investigate the distribution of resistance genes and the clonal relationship amongst imipenem-resistant Acinetobacter baumannii isolates from ten hospitals in Western China as well as to compare the molecular epidemiological data with those of isolates from two hospitals in Hangzhou and Beijing. Genes encoding OXA carbapenemases, metallo-β-lactamases, AmpC cephalosporinase and carbapenem resistance-associated outer membrane protein (CarO) were screened using polymerase chain reaction (PCR) and sequencing. PCR mapping was performed to determine whether insertion sequence ISAba1 elements preceded OXA carbapenemases and AmpC cephalosporinase. International clonal lineages were identified by sequence type multiplex PCR. Multilocus sequence typing (MLST) was performed to determine the sequence types (STs), and then eBURST algorithm was applied to assign clonal complexes (CCs). In this study, dissemination of acquired ISAba1 preceding the bla_OXA-23-like gene was the predominant enzymatic resistance mechanism amongst 272 imipenem-resistant isolates. Five isolates harboured the carO gene disrupted by insertion of ISAba1 and three isolates lacked carO. All of the 36 representative isolates belonged to European clone II. Ten STs, including three novel types, were identified. These STs were clustered into CC92 and two distinct singletons. These observations suggest that imipenem-resistant A. baumannii of European clone II, which carries acquired ISAba1 preceding the bla_OXA-23-like gene and belongs to CC92, has spread within Western China.     

Hospital sewage water: a reservoir for variants of New Delhi metallo-β-lactamase (NDM)- and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae

New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have become a threat to public health. Hospital sewage is generally unexplored, having the potential to harbour bacteria causing healthcare-associated infections. Hence, this study was initiated to monitor NDM-producing Enterobacteriaceae in hospital sewage water. A total of 32 isolates with bla_NDM variants were detected in hospital sewage water, including 17 Escherichia coli, 8 Citrobacter freundii, 4 Shigella boydii, 2 Citrobacter braakii and 1 Citrobacter farmeri, showing resistance to all antibiotics except colistin. All 32 isolates carried bla_NDM (9 bla_NDM-1, 11 bla_NDM-4, 10 bla_NDM-5 and 2 bla_NDM-7), 24 isolates carried bla_CMY variants (1 bla_CMY-2, 3 bla_CMY-4, 5 bla_CMY-6, 11 bla_CMY-42, 2 bla_CMY-86 and 2 bla_CMY-139), 20 isolates carried bla_OXA-type (17 bla_OXA-1 and 3 bla_OXA-48), 19 isolates carried bla_CTX-M and 9 isolates carried ampC on conjugative plasmids of IncFIA, IncFIB, IncFIC, IncP, IncY, IncHI1 and IncI1 types. In E. coli, coexistence of bla_NDM-1 with bla_CMY-6 and bla_CMY-139, of bla_NDM-4 with bla_CMY-6, bla_CMY-42 and bla_CMY-86, of bla_NDM-5 with bla_CMY-6 and bla_CMY-42, and of bla_NDM-7 with bla_CMY-6 was observed. NDM-5-producing S. boydii and NDM-7-producing C. freundii were identified as well as detection of an association of bla_NDM-4 and bla_OXA-48 in C. braakii and C. farmeri. A class 1 integron was also found on a plasmid. ISAba125 and bleomycin genes were found surrounding all bla_NDM variants. The emergence and dissemination of bla_NDM variants in hospital sewage water is a matter of concern, creating an endemic scenario leading to the level of an outbreak.     

Acinetobacter baumannii analysis by core genome multi-locus sequence typing in two hospitals in Bolivia: endemicity of international clone 7 isolates (CC25)

In total, 95 Acinetobacter baumannii isolates recovered from patients from two hospitals in Cochabamba, Bolivia were studied. The presence of class D and B β-lactamases was investigated using polymerase chain reaction, and antimicrobial susceptibility testing was performed by agar dilution and broth microdilution. The resistance rate to carbapenems was 53.7%. All carbapenem-resistant A. baumannii (CRAb, n=51) and four carbapenem-susceptible isolates were further analysed by whole-genome sequencing. The resulting genome assemblies were used to identify the acquired resistome, and core genome multi-locus sequence typing (cgMLST) was used to determine their molecular epidemiology. All but one of the CRAb isolates (n=50) belonged to international clone (IC) 7 and they clustered into five sequence types; on cgMLST, they were found to be separated by ≥40 alleles. All CRAb isolates carried bla_OXA-23 on transposon Tn2008. Metallo-β-lactamases were not detected. These data show that dissemination of several IC7 A. baumannii clones harbouring the carbapenem resistance determinant bla_OXA-23 is occurring in these two hospitals in Cochambamba.     

不同碳青霉烯酶酶型肠杆菌科细菌感染的治疗策略研究

摘要：目的 探讨表达不同碳青霉烯酶的耐碳青霉烯类肠杆菌科细菌(CRE)治疗策略,为临床有效治疗CRE感染提供依据。方法 回顾性分析我院2016年1月—2018年12月临床标本中分离的CRE的相关资料及药敏数据。复苏碳青霉烯耐药的肺炎克雷伯菌、大肠埃希菌和弗氏柠檬酸杆菌,PCR检测其携带的碳青霉烯耐药基因,并比较碳青霉烯酶表型试验与基因结果的一致性。采用肉汤稀释法检测替加环素和头孢他啶/阿维巴坦的敏感性,K-B法检测氨曲南和头孢他啶/阿维巴坦联合作用效果。结果 CRE对常用抗生素具有较高的耐药性。128株CRE菌株均含碳青霉烯耐药基因,其中81株含blaKPC,37株含blaNDM,5株含blaIMP,5株同时含blaKPC和blaNDM。酶抑制剂增强试验能够准确检测各种碳青霉烯酶酶型。替加环素的敏感率为97.6%,头孢他啶/阿维巴坦对产丝氨酸酶菌株的敏感率为100%,而对产金属酶和双酶菌株无效。氨曲南和头孢他啶/阿维巴坦有协同作用,对产金属酶和双酶菌株有很强的抗菌活性。结论 不同酶型CRE可考虑采用不同治疗策略,利用酶抑制剂增强试验确定酶型后,合理选择抗生素进行有效治疗。     

Wide dissemination of OXA-type carbapenemases in clinical Acinetobacter spp. isolates from South Korea

Carbapenem-resistant Acinetobacter spp. are being increasingly reported worldwide, including in South Korea, where we examined 144 representative isolates collected in a nationwide hospital survey in 2005. Metallo-β-lactamases were detected in only 19.4% of isolates, none of which were Acinetobacter baumannii, whereas 74.3% of isolates (mostly A. baumannii) expressed bla_OXA carbapenemase genes. Among the latter, 47 had bla_OXA-23-like genes and 56 had upregulated bla_OXA-51-like variants, including bla_OXA-66, _-83, _-109 and _-115; bla_OXA-115 was a novel variant, detected in two isolates. bla_OXA-72 (bla_OXA-40-like) was detected in only a single Acinetobacter baylyi isolate, whilst three Acinetobacter calcoaceticus isolates had both bla_VIM-2-like and bla_OXA-58 genes. Pulsed-field gel electrophoresis (PFGE) suggested the spread of A. baumannii clones with OXA carbapenemases within and between hospitals. In conclusion, the recent increase in imipenem-resistant Acinetobacter spp. from South Korea is mostly due to OXA-type carbapenemases.     

Inactivation of mgrB gene regulator and resistance to colistin is becoming endemic in carbapenem-resistant Klebsiella pneumoniae in Greece: A nationwide study from 2014 to 2017

IntroductionIn Greece, the spread of carbapenem-resistant Enterobacteriaceae in humans has led to the reintroduction of colistin as a therapeutic agent. Unfortunately, colistin resistance with different mechanisms has emerged. The present work aims to determine the prevalence of carbapenem and colistin resistance and the corresponding mechanisms in Klebsiella pneumoniae clinical isolates from Greece.MethodsFrom 2014 to 2017, 288 carbapenem-resistant K. pneumoniae clinical strains were gathered from a collection of 973 isolates from eight different hospitals in Greece. Antibiotic susceptibility testing was performed using three different methods. Screening of carbapenem and colistin resistance genes was conducted using polymerase chain reaction (PCR) amplification and sequencing.ResultsAmong the 288 (29.6 %) carbapenem-resistant isolates, 213 (73.9%) were colistin-resistant (minimum inhibitory concentration [MIC] >2 mg/L). The KPC type was the most common carbapenemase gene (116; 40.3%), followed by VIM (41; 14.2%), NDM (33; 11.5%) and OXA-48 (22; 7.6%). Moreover, 44 (15.3%) strains co-produced two types of carbapenemases. No mcr genes were detected for colistin resistance but mutations in chromosomal genes were found. These included inactivation of the mgrB gene for 148 (69.5%) strains, including insertion sequences for 94 (44.1%), nonsense mutations for 4 (1.9%) and missense mutations for 24 (11.3%). Moreover, PCR amplification of mgrB gene was negative for 26 (12.2%) strains. Finally, 65 (30.5%) colistin-resistant strains exhibited a wild-type mgrB, the mechanisms of which remain to be elucidated.ConclusionThis study shows that K. pneumoniae clinical strains in Greece are resistant to both carbapenems and colistin and this is endemic and is likely chromosomally encoded.     

Real-time TaqMan PCR for rapid detection of genes encoding five types of non-metallo- (class A and D) carbapenemases in Enterobacteriaceae

A real-time TaqMan multiplex polymerase chain reaction (PCR) assay was developed to detect genes encoding five types of serine carbapenemases (GES, IMI/NMC, KPC, OXA-48 and SME). The assay was validated using control strains known to produce each of these types of enzyme and was then further assessed by ‘blindly’ testing 59 previously characterised clinical isolates, including 19 with serine (KPC or OXA-48) carbapenemases, 22 with metallo- (IMP, VIM or NDM) carbapenemases, and 18 with carbapenem resistance contingent upon extended-spectrum β-lactamase (ESBL) or AmpC production combined with porin loss. The assay detected and correctly assigned the serine carbapenemases in all five positive control strains and in 19 clinical isolates. No false-positive results were seen for isolates with metallo-enzymes or for those that lacked a carbapenemase. The five serine carbapenemase genotypes could also be distinguished by melt-curve analysis or the molecular size of the amplicons.     

Susceptibility of avian influenza viruses of the N6 subtype to the neuraminidase inhibitor oseltamivir

Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC₅₀ values ranging from <0.01 to 5.0 nM; 10% were from 5.1 to 50.0 nM; and none were >50.0 nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades (P ? 0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC₅₀, 30.0 and 40.0 nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.     

Characteristics of carbapenem-resistant Acinetobacter spp. other than Acinetobacter baumannii in South Korea

Although many studies have been performed on carbapenem-resistant Acinetobacter baumannii, only a few studies have addressed carbapenem resistance in Acinetobacter spp. other than A. baumannii (non-baumannii Acinetobacter). Amongst 287 Acinetobacter spp. isolates from patients with bacteraemia in a South Korean hospital collected between 2003 and 2010, 160 (55.7%) were non-baumannii Acinetobacter spp. Antimicrobial susceptibility testing was performed and the effect of efflux pump inhibitors was examined. Antimicrobial resistance genes were identified and pulsed-field gel electrophoresis (PFGE) analysis was performed. OprD expression was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), and CarO disruption was investigated by PCR. Seventeen non-baumannii Acinetobacter isolates (10.6%) were resistant to imipenem or meropenem, comprising eight Acinetobacter pittii (or Acinetobacter genomospecies 3), four Acinetobacter nosocomialis (or Acinetobacter genomospecies 13TU), two Acinetobacter genomospecies ‘close to 13TU’, two Acinetobacter bereziniae (or Acinetobacter genomospecies 10) and one Acinetobacter genomospecies 16. bla_IMP-1 genes were detected in seven and two carbapenem-resistant A. pittii and A. bereziniae isolates, respectively. PFGE showed that A. pittii isolates carrying bla_IMP-1 belonged to the same clone. In addition, bla_SIM-1 and bla_PER-1 genes were simultaneously identified in two A. nosocomialis isolates. In four isolates (one each of A. pittii, A. nosocomialis, Acinetobacter genomospecies ‘close to 13TU’ and Acinetobacter genomospecies 16), efflux pumps were implicated in the increase in carbapenem minimum inhibitory concentrations. No decreased expression of OprD was identified in any carbapenem-resistant non-baumannii Acinetobacter isolates, and disruption of carO was also not detected. Clonal spread of carbapenem-resistant A. pittii carrying bla_IMP-1, which contributes to a high resistance rate in this species, was identified. The bla_IMP-1 and bla_SIM-1 genes were first identified in A. bereziniae and A. nosocomialis, respectively. Since no carbapenem resistance mechanisms could be identified, further efforts to find the resistance mechanism should be made.