Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.     

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.     

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.     

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.     

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.     

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.     

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.     

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla _OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla _CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla _OXA-48 gene in all four isolates was identical to pKP_OXA-48N1, a bla _OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla _OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla _OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.     

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.     

Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid

The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of bla _NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including bla _KPC, bla _IMP, bla _VIM, bla _OXA-48 and bla _NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the bla _KPC-2 genes, and five bla _NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three bla _NDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the bla _NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around bla _NDM-1 (bla _NDM-1-trpF- dsbC-cutA1-groEL-ΔInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller bla _NDM-1 plasmids contained a common gene environment around bla _NDM-1 (IS5-bla _NDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the bla _NDM-1 gene among the CRE.     

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.     

Within-Host and Population Transmission of bla OXA-48 in K. pneumoniae and E. coli

During a large hospital outbreak of OXA-48 producing bacteria, most K. pneumoniae _OXA-48 isolates were phenotypically resistant to meropenem or imipenem, whereas most E. coli _OXA-48 isolates were phenotypically susceptible to these antibiotics. In the absence of molecular gene-detection E. coli _OXA-48 could remain undetected, facilitating cross-transmission and horizontal gene transfer of bla _OXA-48. Based on 868 longitudinal molecular microbiological screening results from patients carrying K. pneumoniae _OXA-48 (n = 24), E. coli _OXA-48 (n = 17), or both (n = 40) and mathematical modelling we determined mean durations of colonisation (278 and 225 days for K. pneumoniae _OXA-48 and E. coli _OXA-48, respectively), and horizontal gene transfer rates (0.0091/day from K. pneumoniae to E. coli and 0.0015/day vice versa). Based on these findings the maximum effect of horizontal gene transfer of bla _OXA-48 originating from E. coli _OXA-48 on the basic reproduction number (R ₀) is 1.9%, and it is, therefore, unlikely that phenotypically susceptible E. coli _OXA-48 will contribute significantly to the spread of bla _OXA-48.     

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.     

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.     

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.     

Molecular Analysis of Acinetobacter baumannii Strains Isolated in Lebanon Using Four Different Typing Methods

This study analyzed 42 Acinetobacter baumannii strains collected between 2009–2012 from different hospitals in Beyrouth and North Lebanon to better understand the epidemiology and carbapenem resistance mechanisms in our collection and to compare the robustness of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), repetitive sequence-based PCR (rep-PCR) and bla _OXA-51 sequence-based typing (SBT). Among 31 carbapenem resistant strains, we have detected three carbapenem resistance genes: 28 carried the bla _OXA-23 gene, 1 the bla _OXA-24 gene and 2 strains the bla _OXA-58 gene. This is the first detection of bla _OXA-23 and bla _OXA-24 in Lebanon. PFGE identified 11 types and was the most discriminating technique followed by rep-PCR (9 types), bla _OXA-51 SBT (8 types) and MLST (7 types). The PFGE type A''/ST2 was the dominant genotype in our collection present in Beyrouth and North Lebanon. The clustering agreement between all techniques was measured by adjust Wallace coefficient. An overall agreement has been demonstrated. High values of adjust Wallace coefficient were found with followed combinations: PFGE to predict MLST types  = 100%, PFGE to predict bla _OXA-51 SBT = 100%, bla _OXA-51 SBT to predict MLST = 100%, MLST to predict bla _OXA-51 SBT = 84.7%, rep-PCR to predict MLST = 81.5%, PFGE to predict rep-PCR = 69% and rep-PCR to predict bla _OXA-51 SBT = 67.2%. PFGE and MLST are gold standard methods for outbreaks investigation and population structure studies respectively. Otherwise, these two techniques are technically, time and cost demanding. We recommend the use of bla _OXA-51 SBT as first typing method to screen isolates and assign them to their corresponding clonal lineages. Repetitive sequence-based PCR is a rapid tool to access outbreaks but careful interpretation of results must be always performed.     

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.     

Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil

The aim of the present study was to investigate the resistance profile, to detect the presence of beta-lactam resistance genes, phenotypic expression of efflux pump systems and class 1 integrons in Pseudomonas spp. strains obtained from untreated hospital effluents. Effluent samples were collected from four hospitals in Porto Alegre, RS, Brazil. Pseudomonas were isolated on MacConkey agar plates and the identification was confirmed by 16S rRNA PCR and biochemical tests. Susceptibility testing was determined by disk-diffusion method using 11 different beta-lactams and MIC assays were performed on isolates resistant to imipenem and ceftazidime. The beta-lactamase genes bla _IMP, bla _VIM, bla _SPM-1, bla _OXA-23-like, bla _OXA-24-like, bla _OXA-51-like and the intl1 gene from class 1 integron were analysed by PCR. One hundred and twenty-four isolates were recovered and the most common species was Pseudomonas pseudoalcaligenes. The resistance found among the isolates was considered high, 62 (50%) isolates were multiresistant. No isolate carrying the beta-lactamase genes tested was found among the strains. Seven isolates showed reduction of MIC for imipenem and ceftazidime in the presence of cyanide m-chlorophenylhydrazone, indicating the hyper expression of efflux pumps. From the 124 isolates, 52 (41.9%) were identified as carrying the class 1 integron gene, intI1. Untreated hospital effluents could be a source of environmental contamination due to discharge of antimicrobial resistant bacteria which can carry integron class 1 and act as a reservoir of resistance genes and have efflux pump systems.