Rapid Molecular Characterization of Acinetobacter baumannii Clones with rep-PCR and Evaluation of Carbapenemase Genes by New Multiplex PCR in Hospital District of Helsinki and Uusimaa

Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab) to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.     

The Carbapenem Inactivation Method (CIM), a Simple and Low-Cost Alternative for the Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative Rods

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.     

Transition of bla OXA-58-like to bla OXA-23-like in Acinetobacter baumannii Clinical Isolates in Southern China: An 8-Year Study

Background

The prevalence of carbapenem-resistant Acinetobacter baumannii in hospitals has been increasing worldwide. This study aims to investigate the carbapenemase genes and the clonal relatedness among A. baumannii clinical isolates in a Chinese hospital.

Methods

Carbapenemase genes and the upstream locations of insertion sequences were detected by polymerase chain reaction (PCR), and the clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.

Results

A total of 231 nonduplicate carbapenemase gene-harboring A. baumannii clinical isolates recovered from Shenzhen People’s Hospital, were investigated between 2002 and 2009. bla _OXA-23-like, bla _OXA-58-like, bla _OXA-40-like, and ISAba1-bla _OXA-51-like were identified in 119, 107, 1, and 4 isolates, respectively. IS1008-ΔISAba3, ISAba3, and ISAba1 were detected upstream of the bla _OXA-58-like gene in 69, 35, and 3 isolates, respectively. All bla _OXA-23-like genes but one had an upstream insertion of ISAba1. bla _OXA-58-like was the most common carbapenemase gene in A.baumannii before 2008, thereafter bla _OXA-23-like became rapidly prevalent and replaced bla _OXA-58-like in 2009. The majority of bla _OXA-58-like-carrying isolates showed lower level of resistance to imipenem and meropenem (minimum inhibitory concentrations (MICs), 1 μg/ml to 16 μg/ml), compared with the majority of bla _OXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/ml for both imipenem and meropenem). All 231 bla _OXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A–N), and three dominant clones A, J, and H accounted for 43.3%, 42.0%, and 8.2% of the tested isolates, respectively. Clone A (sequence type ST92/ST208) with bla _OXA-58-like was the most prevalent before 2008. Clone H (ST229) with bla _OXA-23-like became striking between 2007 and 2008. Clone J (ST381) with bla _OXA-23-like rapidly spread and replaced clones A and H in 2009.

Conclusion

This study is the first to reveal that the distinct bla _OXA-23-like-carrying A. baumannii ST381 displaced the previously prevalent bla _OXA-58-like-carrying A. baumannii ST92/ST208, resulting in the rapidly increasing resistance to carbapenems in A. baumannii in Shenzhen People’s Hospital in 2009.     

First detection of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates in Bulgaria

This report describes the first identification of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates from Bulgaria. According to national surveillance data A. baumannii along with Pseudomonas aeruginosa are the most troublesome microorganisms in hospital environment with high rates of acquired carbapenem resistance. In the present study real-time multiplex PCR was performed to identify the most common carbapenemase genes in 15 non-duplicate carbapenem-resistant A. baumannii isolates collected in 2012. The results showed lack of KPC, GES, VIM, IMP-type enzymes. Four A. baumannii isolates tested positive by PCR for the acquired OXA-24 together with the intrinsic OXA-51 carbapenemase. OXA-24 and OXA-23 were determined as co-existent in one isolate. Two isolates were identified with OXA-23 in addition to the OXA-51 carbapenemase.     

Molecular Characteristics of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae from Humans in the Community

Objective

To investigate the molecular characteristics of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae collected during a cross-sectional study examining the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in humans living in areas with high or low broiler density.

Methods

ESC-resistant Enterobacteriaceae were identified by combination disc-diffusion test. ESBL/AmpC/carbapenemase genes were analysed using PCR and sequencing. For E. coli, phylogenetic groups and MLST were determined. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing.

Results

175 ESC-resistant Enterobacteriaceae were cultured from 165/1,033 individuals. The isolates were Escherichia coli(n=65), Citrobacter freundii (n=52), Enterobacter cloacae (n=38), Morganella morganii (n=5), Enterobacter aerogenes (n=4), Klebsiella pneumoniae (n=3), Hafnia alvei (n=2), Shigella spp. (n=2), Citrobacter amalonaticus (n=1), Escherichia hermannii (n=1), Kluyvera cryocrescens (n=1), and Pantoea agglomerans (n=1). The following ESBL genes were recovered in 55 isolates originating from 49 of 1,033 (4.7 %) persons: bla _CTX-M-1 (n=17), bla _CTX-M-15 (n=16), bla _CTX-M-14 (n=9), bla _CTX-M-2 (n=3), bla _CTX-M-3 (n=2), bla _CTX-M-24 (n=2), bla _CTX-M-27 (n=1), bla _CTX-M-32 (n=1), bla _SHV-12 (n=2), bla _SHV-65 (n=1) and bla _TEM-52 (n=1). Plasmidic AmpC (pAmpC) genes were discovered in 6 out of 1,033 (0.6 %) persons. One person carried two different E. coli isolates, one with bla _CTX-M-1 and the other with bla _CMY-2 and therefore the prevalence of persons carrying Enterobacteriaceae harboring ESBL and/or pAmpC genes was 5.2 %. In eight E. coli isolates the AmpC phenotype was caused by mutations in the AmpC promoter region. No carbapenemase genes were identified. A large variety of E. coli genotypes was found, ST131 and ST10 being most common.

Conclusions

ESBL/pAmpC genes resembled those from patients in Dutch hospitals, indicating that healthy humans form a reservoir for transmission of these determinants to vulnerable people. The role of poultry in the transmission to humans in the community remains to be elucidated.     

鲍曼不动杆菌的耐药情况及碳青霉烯酶基因检测

了解佳木斯大学附属第一医院鲍曼不动杆菌耐药性及碳青霉烯酶包括苯唑西林酶和金属酶相关耐药基因分布情况,为临床抗菌药物的合理选择提供依据。2013年9月至2014年12月使用VITEK-II全自动微生物鉴定/药敏测试系统筛选出佳木斯大学附属第一医院临床标本鲍曼不动杆菌69株;采用多重PCR方法检测鲍曼不动杆菌携带的碳青霉烯酶相关耐药基因16SrRNA、OXA-23、OXA-24、OXA-51、OXA-58、IMP、VIM、SIM,并对耐药基因扩增的阳性产物进行DNA 序列分析。69株AB对亚胺培南、美洛培南的耐药率分别为36.2%、37.68%,对其他抗菌药物的耐药率均高于50%。6种耐药基因的检测结果为69株(100%)携带OXA-51基因,32株(46.4%)携带OXA-23基因,17株(24.6%)携带OXA-24基因,5株(7.2%)携带OXA 58基因,1株（1.4%）携带IMP基因。25株碳青霉烯类药物耐药鲍曼不动杆菌中,22株(88%) 携带OXA-23,1株(4%)携带OXA-58,10株(40%)携带OXA-24,6株（24%）同时携带OXA-23、OXA-24。 DNA序列分析结果显示：OXA-23、OXA-24、OXA-51、OXA-58分别与NCBI的序列同源性均为99%。产OXA-23型碳青霉烯酶可能是佳木斯大学附属第一医院鲍曼不动杆菌对碳青霉烯酶类抗菌药物耐药的主要原因,另外佳木斯大学附属第一医院存在OXA-24型耐药基因鲍曼不动杆菌的区域性流行。     

Prevalence and Characterization of Carbapenem-Resistant Enterobacteriaceae Isolated from Mulago National Referral Hospital,Uganda

Introduction

Carbapenemases have increasingly been reported in enterobacteriaceae worldwide. Most carbapenemases are plasmid encoded hence resistance can easily spread. Carbapenem-resistant enterobacteriaceae are reported to cause mortality in up to 50% of patients who acquire bloodstream infections. We set out to determine the burden of carbapenem resistance as well as establish genes encoding for carbapenemases in enterobacteriaceae clinical isolates obtained from Mulago National Referral Hospital, Uganda.

Methods

This was a cross-sectional study with a total of 196 clinical isolates previously collected from pus swabs, urine, blood, sputum, tracheal aspirates, cervical swabs, endomentrial aspirates, rectal swabs, Vaginal swabs, ear swabs, products of conception, wound biopsy and amniotic fluid. All isolates were subjected to phenotypic carbapenemase screening using Boronic acid-based inhibition, Modified Hodge and EDTA double combined disk test. In addition, all the isolates were subjected to PCR assay to confirm presence of carbapenemase encoding genes.

Results

The study found carbapenemase prevalence of 22.4% (44/196) in the isolates using phenotypic tests, with the genotypic prevalence slightly higher at 28.6% (56/196). Over all, the most prevalent gene was blaVIM (21,10.7%), followed by blaOXA-48 (19, 9.7%), blaIMP (12, 6.1%), blaKPC (10, 5.1%) and blaNDM-1 (5, 2.6%). Among 56 isolates positive for 67 carbapenemase encoding genes, Klebsiella pneumonia was the species with the highest number (52.2%). Most 32/67(47.7%) of these resistance genes were in bacteria isolated from pus swabs.

Conclusion

There is a high prevalence of carbapenemases and carbapenem-resistance encoding genes among third generation cephalosporins resistant Enterobacteriaceae in Uganda, indicating a danger of limited treatment options in this setting in the near future.     

Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A,B, and D

Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.     

Co-occurrence of genes encoding carbapenemase,ESBL, pAmpC and non-β-Lactam resistance among Klebsiella pneumonia and E. coli clinical isolates in Tunisia

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried bla_NDM-1 (n = 11), bla_OXA-48 (n = 11), bla_NDM-1 + bla_OXA-48 (n = 1), bla_NDM-1 + bla_VIM-1 (n = 1), bla_OXA-204 (n = 1), along with bla_CTX-M, bla_OXA, bla_TEM, bla_CMY, bla_DHA and bla_SHV genes variants on conjugative plasmid of IncL/M, IncR, IncFII_K, IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.     

Characterization of a Novel Plasmid Type and Various Genetic Contexts of bla OXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/Objective

Several studies have described the epidemiological distribution of bla _OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla _OXA-58-carrying plasmids and the genetic context surrounding bla _OXA-58 in Acinetobacter spp. in China.

Methodology/Principal Findings

Twelve non-duplicated bla _OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla _OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla _OXA-58-harboring plasmids. The genetic structure surrounding bla _OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla _OXA-58 and bla _OXA-23. bla _OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla _OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla _OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla _OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla _OXA-58 were carbapenem susceptible.

Conclusion

The study revealed the unique features of bla _OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla _OXA-58 contributed to various antibiotics resistance profiles.     

High occurrence of carbapenem-resistant Escherichia coli isolates from healthy rabbits (Oryctolagus cuniculus): first report of blaIMI and blaVIM type genes from livestock in Tunisia

We aimed to study the antibiotic susceptibility and possible occurrence of extended-spectrum beta-lactamases (ESBL)/carbapenemase-producing Escherichia coli isolates collected from rabbits in Tunisia. In all, 35 faecal samples from healthy rabbits were collected from one farm and E. coli were isolated from three media: antibiotic-free TBX agar, TBX+2 mg l⁻¹ cefotaxime and TBX+1 mg l⁻¹ imipenem. In total, 39 E. coli isolates were recovered; the majority showed resistance to at least one antibiotic and none was ESBL producer. Carbapenem resistance was detected in 16 isolates from either selective or un-selective media. Phenotypic methods used to detect carbapenemase production showed two positive isolates by Modified Hodge Test, six metallo-carbapenemase producers (Imipenem disc+EDTA) and all were temocillin resistant (possible OXA-48 carbapenemase). bla_VIM and bla_IMP type genes were detected in two and one isolates, respectively; one of them harboured both genes. Isolates contained common genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tetA, tetB, tetC) and fluoroquinolones (qnrS, aac(6′)-Ib-cr). Class 1 and 2 integrons were detected in five and four isolates, respectively. These findings highlight the importance of rabbit production as reservoir of carbapenem-resistant E. coli and argument the first report of bla_VIM and bla_IMP genes in livestock in Tunisia.     

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla _OXA-23 was the most frequently detected (82%), followed by bla _OXA-24 (11%) and bla _OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.     

Carbapenem Non-Susceptible Enterobacteriaceae in Quebec,Canada: Results of a Laboratory Surveillance Program (2010–2012)

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) represent a major public health concern because these bacteria are usually extensively resistant to most antibiotics. In order to evaluate their dissemination in Quebec, a surveillance program was introduced in 2010. We report the molecular and epidemiological profiles of CPE isolates collected. Between August 2010 and December 2012, a total of 742 non-duplicate isolates non-susceptible to carbapenems were analysed. AmpC β-lactamase and metallo-β-lactamase production were detected by Etest and carbapenemase production by the modified Hodge test (MHT). Antibiotic susceptibility profiles were determined using broth microdilution or Etest. Clonality of Klebsiella pneumoniae carbapenemase (KPC) strains was analyzed by pulsed-field gel electrophoresis (PFGE). The presence of genes encoding carbapenemases as well as other β-lactamases was detected using PCR. Of the 742 isolates tested, 169 (22.8%) were CPE. Of these 169 isolates, 151 (89.3%) harboured a bla _KPC gene while the remaining isolates carried bla _SME (n = 9), bla _OXA-48 (n = 5), bla _NDM (n = 3), and bla _NMC (n = 1) genes. Among the 93 KPC strains presenting with a unique pattern (unique PFGE pattern and/or unique antibiotics susceptibility profile), 99% were resistant to ertapenem, 95% to imipenem, 87% to meropenem, 97% to aztreonam, 31% to colistin and 2% to tigecycline. In 19 patients, 2 to 5 KPC strains from different species or with a different PFGE pattern were isolated. CPE strains were present in the province of Quebec with the majority of strains harbouring KPC. Alternately, SME, OXA-48 and NMC containing strains were rarely found.     

Genetic Characterization of Carbapenem-Resistant Enterobacteriaceae and the Spread of Carbapenem-Resistant Klebsiella pneumonia ST340 at a University Hospital in Thailand

Carbapenem-resistant Enterobacteriaceae (CRE) has increasingly spread worldwide in the past decade. The prevalence and characteristics of CRE in Thailand are unknown. In this study, we conducted a 2-year surveillance of CRE among 12,741 clinical isolates of Enterobacteriaceae at the largest university hospital in Thailand with molecular characterization of beta-lactamase (bla) genes, including carbapenemase genes. The CRE prevalence was 1.4%. bla _KPC-13 and bla _IMP-14a were the only carbapenemase genes detected among these CRE isolates. bla _KPC-13 gene was found in a single isolate of Escherichia coli, Enterobacter cloacae and Citrobacter freundii, and bla _IMP-14a was found in four isolates of Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) isolates were resistant to multiple carbapenems at a higher ratio than other CRE species, and thus were further characterized for resistance phenotypes, bla genotypes and molecular epidemiology. Most CRKP isolates harboured multiple bla genes, especially those related to extended-spectrum beta-lactamases. Seven CRKP isolates were resistant to all tested carbapenems, and showed decreased ompK35 and/or ompK36 porin gene expression. Molecular typing of CRKP based on pulsed-field gel electrophoresis (PFGE) demonstrated several unrelated clones. Multilocus sequence typing (MLST) was partially concordant with PFGE results and revealed that ST340, a member of drug-resistant K. pneumoniae clonal complex 258, was the most predominant clone, followed by ST48, ST11 and ST273. The novel ST1645 was identified from this study. ST340 has neither been shown to be predominated among CRKP from other studies, nor been reported in Thailand. Therefore, it emphases a critical concern to monitor and control the spread of CRKP.     

Clonal Dissemination of KPC-2, VIM-1, OXA-48-Producing Klebsiella pneumoniae ST147 in Katowice,Poland

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important bacterium of nosocomial infections. In this study, CRKP strains, which were mainly isolated from fecal samples of 14 patients in three wards of the hospital in the Silesia Voivodship, rapidly increased from February to August 2018. Therefore, we conducted microbiological and molecular studies of the CRKP isolates analyzed. Colonized patients had critical underlying diseases and comorbidities; one developed bloodstream infection, and five died (33.3%). Antibiotic susceptibilities were determined by the E-test method. A disc synergy test confirmed carbapenemase production. CTX-Mplex PCR evaluated the presence of resistance genes bla_CTX-M-type, bla_CTX-M-1, bla_CTX-M-9, and the genes bla_SHV, bla_TEM, bla_KPC-2, bla_NDM-1, bla_OXA-48, bla_IMP, and bla_VIM-1 was detected with the PCR method. Clonality was evaluated by Multi Locus Sequence Typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Six (40%) strains were of XDR (Extensively Drug-Resistant) phenotype, and nine (60%) of the isolates exhibited MDR (Multidrug-Resistant) phenotype. The range of carbapenem minimal inhibitory concentrations (MICs, μg/mL) was as follows doripenem (16 to >32), ertapenem (> 32), imipenem (4 to > 32), and meropenem (> 32). PCR and sequencing confirmed the bla_CTX-M-15, bla_KPC-2, bla_OXA-48, and bla_VIM-1 genes in all strains. The isolates formed one large PFGE cluster (clone A). MLST assigned them to the emerging high-risk clone of ST147 (CC147) pandemic lineage harboring the bla_OXA-48 gene. This study showed that the K. pneumoniae isolates detected in the multi-profile medical centre in Katowice represented a single strain of the microorganism spreading in the hospital environment.     

Evaluation of an Automated Rapid Diagnostic Assay for Detection of Gram-Negative Bacteria and Their Drug-Resistance Genes in Positive Blood Cultures

We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla _CTX-M, 119 bla _IMP, 8 bla _KPC, 16 bla _NDM, 24 bla _OXA-23, 1 bla _OXA-24/40, 1 bla _OXA-48, 4 bla _OXA-58, and 6 bla _VIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.     

Characterization of Carbapenem-Resistant Enterobacteriaceae with High Rate of Autochthonous Transmission in the Arabian Peninsula

To establish the role of local transmission versus possible pathogen import due to previous foreign exposure in infections caused by carbapenem non-susceptible Enterobacteriaceae in the Arabian Peninsula, 200 independent isolates collected in 16 hospitals of Saudi Arabia, Kuwait, Oman and the United Arab Emirates were studied. All strains were multidrug resistant; 42.5% of them also qualified as extremely drug resistant. The frequency of various carbapenemases varied according to the participating countries, but in the collection, as a whole, bla _NDM-1 was the most frequently encountered carbapenemase gene (46.5%) followed by bla _OXA-48-like gene (32.5%). A comparatively high rate (8.9%) of multi-clonal strains carrying both bla _NDM and bla _OXA-48-like genes in the United Arab Emirates, representing the most resistant subgroup, was encountered. No KPC-expressing isolates were detected. Three major clones of bla _NDM-1 carrying Klebsiella pneumoniae of ST152 (n = 22, Saudi Arabia), ST14 (n = 7, United Arab Emirates) and ST147 types (n = 9, Oman) were identified, the latter two clones carrying similar, but not identical HI1b incompatibility type plasmids of >170kb. While from 78.6% of the cases with documented foreign hospitalization bla _NDM positive strains were isolated, these strains formed only 25.6% of all the isolates expressing this enzyme. In fact, 56.8% of the NDM, 75.7% of OXA-48-like and 90.9% of VIM positive strains were recovered from patients without documented foreign exposure, neither in the form of travel or prior hospitalization abroad, suggesting a high rate of autochthonous infections. This, considering the extensive links of these countries to the rest of the world, predicts that trends in the local epidemiology of carbapenem resistant strains may increasingly affect the spread of these pathogens on the global scale. These results call for improved surveillance of carbapenem resistant Enterobacteriaceae in the countries of the Arabian Peninsula.     

Molecular Characterization of Klebsiella pneumoniae Carbapenemase (KPC)-Producing Enterobacteriaceae in Ontario,Canada, 2008-2011

Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of bla _KPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as bla _KPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored bla _KPC-2 (n = 23) or bla _KPC-3 (n = 7). bla _TEM-1 (n = 27) was commonly detected and occasionally bla _OXA-1 (n = 3) and bla _CTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried bla _SHV-11. bla _KPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258.