High-level Carbapenem Resistance Research Articles

Coexistence of blaKPC-2 and blaNDM-1 in one IncHI5 plasmid confers transferable carbapenem resistance from a clinical isolate of Klebsiella michiganensis in China

This study firstly identified an IncHI5 plasmid pK254-KPC_NDM co-carrying two different class carbapenemase genes blaKPC-2 and blaNDM-1 in Klebsiella michiganensis K254. The strain K254 was sequenced by high-throughput genome sequencing. A detailed genomic and phenotypic characterization of pK254-KPC_NDM was performed. pK254-KPC_NDM displayed the conserve IncHI5 backbone and carried a resistant accessory region: Tn1696-related transposon Tn7414 containing blaKPC-2 and blaNDM-1. A sequence comparison was applied to a collection of four Tn1696-related transposons (Tn7414-Tn7417) harbouring carbapenemase genes. For all these four transposons, the blaNDM-1 was carried by Tn125 derivatives within three different mobile genetic elements. Tn7414 further acquired another carbapenemase gene, blaKPC-2, because of the integration of the local blaKPC-2 genetic environment from Tn6296, resulting in the high-level carbapenem resistance of K. michiganensis K254. The conjugal transfer and plasmid stability experiments confirmed that pK254-KPC_NDM could be transferred intercellularly and keep the stable vertical inheritance in different bacteria, which would contribute to the further dissemination of multiple carbapenemase genes and enhance the adaption and survival of K. michiganensis under complex and diverse antimicrobial selection pressures. This study was the first to report the K. michiganensis isolate coharbouring blaKPC-2 and blaNDM-1 in the Tn1696-related transposon in IncHI5 plasmid. The emergence of novel transposons simultaneously carrying multiple carbapenemase genes might contribute to the further dissemination of high-level carbapenem resistance in the isolates of the hospital settings and pose new challenges for the treatment of nosocomial infection.

Recurrent Campylobacter jejuni Infections with In Vivo Selection of Resistance to Macrolides and Carbapenems: Molecular Characterization of Resistance Determinants.

We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects β-strands 5 and 6 and forms a constriction zone involved in Ca2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA's extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within porA. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp.

Characterization difference of typical KL1, KL2 and ST11-KL64 hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

Almost all the formation of hypervirulent and carbapenem-resistant Klebsiella pneumoniae follow two major patterns: KL1/KL2 hvKP strains acquire carbapenem-resistance plasmids (CR-hvKP), and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids (hv-CRKP). These two patterns may pose different phenotypes. In this study, three typical resistance and hypervirulent K. pneumoniae (KL1, KL2, and ST11-KL64), isolating from poor prognosis patients, were selected. Compared with ST11-KL64 hv-CRKP, KL1/KL2 hypervirulent lineages harbor significantly fewer resistance determinants and exhibited lower-level resistance to antibiotics. Notably, though the blaKPC gene could be detected in all these isolates, KL1/KL2 hvKP strain did not exhibit corresponding high-level carbapenem resistance. Unlike the resistance features, we did not observe significant virulence differences between the three strains. The ST11-KL64 hv-CRKP (1403) in this study, showed similar mucoviscosity, siderophores production, and biofilm production compared with KL1 and KL2 hvKP. Moreover, the hypervirulent of ST11-KL64 hvKP also verified with the human lung epithelial cells infection and G. mellonella infection models. Moreover, we found the pLVPK-like virulence plasmid and IncF blaKPC-2 plasmid was crucial for the formation of hypervirulent and carbapenem-resistant K. pneumoniae. The conservation of origin of transfer site (oriT) in these virulence and blaKPC-2 plasmids, indicated the virulence plasmids could transfer to CRKP with the help of blaKPC-2 plasmids. The co-existence of virulence plasmid and blaKPC-2 plasmid facilitate the formation of ST11-KL64 hv-CPKP, which then become nosocomial epidemic under the antibiotic stress. The ST11-KL64 hv-CPKP may poses a substantial threat to healthcare networks, urgent measures were needed to prevent further dissemination in nosocomial settings.

Whole-Genome Analysis of blaNDM-Bearing Proteus mirabilis Isolates and mcr-1-Positive Escherichia coli Isolates Carrying blaNDM from the Same Fresh Vegetables in China.

The global spread of colistin or carbapenem-resistant Enterobacteriaceae (CRE) has been a pressing threat to public health. Members of Enterobacteriaceae, especially Proteus mirabilis and Escherichia coli, have been prevalent foodborne pathogens and such pathogens from fresh vegetables have triggered foodborne illness in China. However, reports about CRE, especially P. mirabilis from fresh vegetables, are still lacking. In this study, we identified five blaNDM-positive P. mirabilis and five blaNDM-positive generic E. coli concurrently from five fresh vegetables in two markets from China, and four of the five E. coli also carried mcr-1. The 10 isolates were characterized with methods including antimicrobial susceptibility testing, conjugation, whole-genome sequencing and phylogenetic analysis. All 10 isolates were multidrug-resistant (MDR). blaNDM-5 in five E. coli isolates and one P. mirabilis carrying blaNDM-5 was located on similarly transferable IncX3 plasmids, while transferably untypable plasmids were the carriers of blaNDM-1 in four P. mirabilis isolates from different types of vegetables/markets. mcr-1 in the four blaNDM-5-positive E. coli was located on similarly non-conjugative IncHI2 MDR plasmids lacking transfer region. Notably, ISCR1 complex class 1 integron capable of capturing blaNDM-1 was found on all untypable plasmids from P. mirabilis, and five copies of ISCR1 complex class 1 integron containing blaNDM-1 even occurred in one P. mirabilis, which showed high-level carbapenem resistance. Plasmid and phylogenetic analysis revealed that the blaNDM-positive P. mirabilis and E. coli from fresh vegetables might be derived from animals and transmitted to humans via the food chain. The concurrence of blaNDM-positive P. mirabilis and E. coli carrying both mcr-1 and blaNDM in different types of fresh vegetables eaten raw is alarming and threatens food safety. Sustained surveillance of these foodborne pathogens among fresh vegetables is urgent to ensure the health of food consumers. We report for the first time the concurrence of blaNDM-positive P. mirabilis and mcr-1-bearing E. coli carrying blaNDM from the same fresh vegetables.

Description of a Rare Pyomelanin-Producing Carbapenem-Resistant Acinetobacter baumannii Strain Coharboring Chromosomal OXA-23 and NDM-1.

ABSTRACTCarbapenem-resistant Acinetobacter baumannii (CRAB), which belonged to global clones 1 (GC1) or 2 (GC2), has been widely reported and become a global threat. However, non-GC1 and non-GC2 CRAB strains are not well-studied, especially for those with rare phenotype. Here, one pyomelanin-producing CRAB strain (A. baumannii DETAB-R21) was isolated from oral swab in the ICU. Antimicrobial susceptibility testing showed it was resistant to carbapenems, ceftazidime, levofloxacin, and ciprofloxacin. DETAB-R21 was ST164Pas and ST1418Oxf with KL47 and OCL5, respectively. Whole-genome sequencing (WGS) analysis revealed chromosome contained three copies of blaOXA-23 on three 4,805-bp Tn2006 composite transposons with various novel 9-bp target site duplications (TSD). A Tn125-like structure, including blaNDM-1, a novel 4,343 bp composite transposon encoding blaCARB-16, and three prophage regions were also identified. Importantly, hmgA was interrupted by a Tn2006 and contributed to pyomelanin production and further confirmed by hmgA overexpression. Furthermore, A. baumannii irradiated with UV light, DETAB-R21 showed a higher relatively survival rate compared to a control strain that did not produce pyomelanin. No effects of pyomelanin were observed on disinfectants susceptibility, growth, or virulence. In conclusion, pyomelanin-producing CRAB carrying the blaNDM-1 and blaOXA-23 genes embedded in the bacterial chromosome is of grave concern for health care settings, highlighting the need for effective measures to prevent further dissemination.IMPORTANCE Pyomelanin production is a quite rare phenotype in A. baumannii. Moreover, the mechanisms leading to the pyomelanin production was still unclear. Here, we for the first time, confirmed the mechanism of pyomelanin production, and further investigated the impact of pyomelanin on disinfectants susceptibility, growth, virulence, and UV irradiation. More importantly, many mobile genetic elements (MGEs), including three copies of Tn2006 composite transposons, one copy of blaNDM-1 on the Tn125-like structure and three prophage regions, were identified in the chromosome, demonstrated strong plasticity of A. baumannii genome. Our study provides important insights into the new rare ST164Pas A. baumannii strain with high level carbapenem resistance, which is of great threat for patients. These findings will provide important insights into the resistance gene transfer via transposition events and further spread in the clinic.

Clonal Dissemination of Aeromonas hydrophila With Binary Carriage of bla KPC-2-Bearing Plasmids in a Chinese Hospital.

Dissemination of the Klebsiella pneumoniae carbapenemase (KPC)-encoding gene among Enterobacterales is common but relatively rare in Aeromonas spp. In this study, we characterized two KPC-2-producing Aeromonas hydrophila strains (Ah2101 and Ah2111), each isolated from a patient in different intensive care units (ICUs) of a Chinese hospital. Whole-genome sequencing (WGS) revealed simultaneous carriage of the blaKPC−2 and imiH genes, both of which encode high-level carbapenem resistance in these two A. hydrophila isolates. The two isolates were shown to be clonally related and each isolate harbored two distinguishable blaKPC−2-bearing plasmids, only one of which was transferrable to A. hydrophila, but not Escherichia coli EC600 via conjugation. The genetic element that contains blaKPC−2 in these two plasmids, namely ISKpn27-ΔblaTEM−1-blaKPC−2-ISKpn6, was structurally identical, commonly detected in Enterobacterales, and associated with Tn3-based transposons. In addition, more than sixty putative genes that encode various virulence factors were identified in these two A. hydrophila isolates. This is the first study that reports clonal dissemination of carbapenem-resistant A. hydrophila strains carrying structurally different blaKPC−2-bearing plasmids. Further investigation is warranted to monitor the future transmission of blaKPC−2-bearing plasmids in A. hydrophila in clinical settings.

Bacterial characteristics of carbapenem-resistant Enterobacteriaceae (CRE) colonized strains and their correlation with subsequent infection

BackgroundSearching the risk factors for carbapenem-resistant Enterobacteriaceae (CRE) infection is important in clinical practice. In the present study, we aim to investigate bacterial characteristics of colonizing strains and their correlation with subsequent CRE infection.MethodsBetween May 2018 and January 2019, patients hospitalized in the department of haematology and intensive care unit (ICU) were screened for CRE by rectal swabs and monitored for the outcome of infection. We identified the species and carbapenemase-encoding genes of colonizing strains and performed antimicrobial susceptibility tests and multilocus sequence typing (MLST). Risk factors for subsequent CRE infections were ascertained by univariate and multivariable analysis.ResultsWe collected a total of 219 colonizing strains from 153 patients. Klebsiella pneumoniae was the most abundant species, and MLST analysis showed rich diversity. K. pneumoniae carbapenemase (KPC) was predominant in the infection group (72.4%). In the non-infection group, 35.4% of strains were non-carbapenemase-producing CRE (NCP-CRE), and New Delhi metallo-β-lactamase (NDM) was predominant (42.2%). The rate of high-level carbapenem resistance (minimum inhibitory concentration [MIC] ≥ 64 mg/L for meropenem and ertapenem, ≥ 32 mg/L for imipenem) was remarkably higher in the infection group than in the non-infection group (P < 0.001). Univariate analysis showed that K. pneumoniae, high-level carbapenem resistance, CP-CRE and KPC-CRE were infection risk factors after CRE colonization. On multivariable analysis with different carbapenemase dichotomizations, KPC-CRE (adjusted odds ratio [aOR], 4.507; 95% confidence interval [CI], 1.339–15.171; P = 0.015) or imipenem MIC ≥ 32 mg/L (aOR, 9.515; 95% CI, 1.617–55.977; P = 0.013) were respectively identified as independent risk factors for subsequent infection.ConclusionsPatients colonized with KPC-CRE or strains with an imipenem MIC ≥ 32 mg/L were at particularly high risk of subsequent CRE infections during their hospital stay.

Emergence of High Level Carbapenem and Extensively Drug Resistant Escherichia coli ST746 Producing NDM-5 in Influent of Wastewater Treatment Plant, Seoul, South Korea.

High level carbapenem and extensively drug resistant (XDR) Escherichia coli strain N7, which produces a variant of New Delhi metallo-β-lactamase (NDM-5), was isolated from the influent of the Jungnang wastewater treatment plant located on Han River, Seoul, South Korea. Phenotypic and genotypic resistances to carbapenem were tested using agar and broth dilution methods, and polymerase chain reaction. Whole-genome sequencing was performed to characterize the genetic structure of strain N7. E. coli strain N7, which harbors the blaNDM–5 gene, showed high level of carbapenem resistance at concentrations of doripenem (512 mg/L) and meropenem (256 mg/L), and XDR to 15 antibiotics. Based on the genomic sequence analysis, two plasmids, a hybrid IncHI2/N-type and an IncX3 type, were present. The former contains a cluster (blaNDM–5-bleMBL-trpF-dsbD) bracketed by multi-insertional sequences, IS3000, ISAba125, IS5, and IS26. The latter carries the following resistance genes: blaCTX–14, aac(3)-IV, aadA1, aadA2, aph(3′)-Ia, aph(4)-Ia, sul1, sul2, sul3, dfrA12, fosA3, oqxA, oqxB, mph(A), and floR, and cmlA1. The chromosome, contig3, and contig5 also carry blaCTX–64 and mdf(A), tet(A), and erm(B), tet(M) and aadA22, respectively. Strain N7 also harbors virulence factors such as fimH, flu, ecpABCDE, sfmA, hlyE, and gadA. This study demonstrates the emergence of high level carbapenem resistant XDR E. coli strain N7 containing blaNDM–5 in aquatic environment, Seoul, South Korea. Due to the presence of mobile genetic elements, this strain could horizontally transfer resistance genes, including blaNDM–5 to environmental bacteria. Thus, it is necessary to conduct continuous surveillance for carbapenem resistance in various aquatic environments.

Carbapenemase detection testing in the era of ceftazidime/avibactam-resistant KPC-producing Enterobacterales: A 2-year experience

ObjectivesThe aim of this study was to investigate the prevalence of ceftazidime/avibactam (CZA) resistance among carbapenemase-producing Enterobacterales (CPE) blood culture isolates as well as the performance of the main carbapenemase phenotypic detection methods to identify KPC variants associated with CZA resistance. MethodsNon-duplicate CPE strains isolated from blood cultures during 2018–2020 were tested for antimicrobial susceptibility. Molecular testing was used to identify carbapenemase-producers. Strains harbouring blaKPC and with a CZA minimum inhibitory concentration (MIC) ≥8 mg/L were investigated by sequencing. Subsequentially, five phenotypic carbapenemase detection methods were evaluated on these strains, namely the modified carbapenem inactivation method (mCIM), Rapidec® Carba NP, the disk diffusion synergy test, NG-Test CARBA® 5 and RESIST-5 O.O.K.N.V. ResultsOverall, the CZA resistance rate was high (13.7%) and remained relevant (5.9%) excluding metallo-β-lactamases-producers. All isolates harbouringblaKPC mutants (n = 8) were associated with reduced carbapenem MICs and negative results by all detection methods based on revelation of enzyme activity. Lateral flow immunoassays failed to detect KPC-31 (n = 4) and KPC-33 (n = 2) but correctly identified KPC-14 (n = 2). Conversely, isolates harbouring wild-type KPC genes (n = 3) were associated with high-level CZA resistance and carbapenem resistance and tested positive by all of the evaluated methods. ConclusionIn the era of CZA-based therapies, molecular blaKPC identification followed by a carbapenem hydrolysis-based phenotypic assay could be the most reasonable diagnostic algorithm to detect all KPC-producers and to identify mutants associated with impaired carbapenemase activity and CZA resistance.

Diversity of L1/L2 genes and molecular epidemiology of high-level carbapenem resistance Stenotrophomonas maltophilia isolates from animal production environment in China.

Stenotrophomonas maltophilia is emerging as a significant cause of human and animal disease worldwide. A total of 3400 samples were collected from animal farms and adjacent environments in China. The blaL1 and blaL2 genes were identified using whole genome sequence analyses and examined by phylogenetics. Isolates were also tested for susceptibility to 18 antibiotics. We isolated 118 strains of S. maltophilia from 3400 samples. The positive rates of blaL1 and blaL2 genes were 75% (89/118) and 22% (26/118) and we identified 11 L1 and 6 L2 amino acid sequence variants. S. maltophilia has at least two inducible β-lactamases (L1 and L2) that can hydrolyze almost all classes of β-lactams and these genes are suspected to confer carbapenem resistance. This represents a significant public health threat especially for hospitalized patients. We conducted a molecular surveillance study on the prevalence and characteristics of the blaL1 and blaL2 genes of S. maltophilia.

Carbapenemase Producers Among Extensive Drug-Resistant Gram-Negative Pathogens Recovered from Febrile Neutrophilic Patients in Egypt.

PurposeThis study aimed to detect the prevalence of carbapenemase producers (CPs) among extensive drug-resistant (XDR)-carbapenemase producing Gram-negative bacteria (GNB) recovered from various clinical specimens of hospitalized neutrophilic febrile patients in two major tertiary care hospitals in Egypt.MethodsStandard methods were used to evaluate the antimicrobial susceptibility of clinical isolates according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Phenotypic and genotypic analysis of CPs were carried out and statistically analyzed using standard methods.ResultsThree hundred and forty-two GNB were obtained from 342 clinical specimens during the period of the study, where 162 (47%) were enterobacterial isolates, including, 63 (18.4%) Escherichia coli, 87 (25.4%) Klebsiella spp., 5 (1.46%) Enterobacter cloacae, 5 (1.46%) Salmonella spp. and 2 (0.6%) Proteus and 180 (53%) were non-fermentative bacilli including, 129 (37.7%), Acinetobacter baumannii, and 51 (14.9%), Pseudomonas spp. Out of the 342 GNB, 188 (54.9%) isolates were multi-drug resistant (MDR). Of these, 52 (27.6%) were XDR as well as CPs as confirmed phenotypically. The MIC of imipenem against the XDR GNB against showed either low (11 isolates; 21.1%; MIC range =4–32 µg/mL) or high levels of resistance (41 isolates; 78.8%; MIC range = 64-≥1024). The most prevalent carbapenem resistance (CR) genes were blaKPC (63.5%) followed by blaOXA-48 (55.7%) and blaVIM (28.8%). No significant association could be observed between the MIC level and the presence of CR genes (P value >0.05).ConclusionHigh prevalence of MDR (54.9%) and XDR (27.6%) GNB pathogens associated with high levels of resistance to carbapenems were observed. All XDR GNB were CPs and tested positive for at least one of the CR genes. However, most of them (78.8%) showed a high level of CR (MIC range = 64-≥1024) with no significant association with the CR genes.

Molecular epidemiology and resistance patterns of blaOXA-48Klebsiella pneumoniae and Escherichia coli: A nationwide multicenter study in Taiwan.

We describe the molecular epidemiology and resistance patterns of blaOXA-48Klebsiella pneumoniae and Escherichia coli in Taiwan. In this multicenter surveillance study from January 2012 to August 2015, the identified blaOXA-48Enterobacteriaceae isolates were subjected to antibiotics susceptibility testing. PCR method was used for detecting concomitant other beta-lactamases. Outer membrane porins were analyzed. Genetic relatedness and molecular epidemiology of the isolates were determined through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid incompatibility was determined using PCR-based replicon typing. Forty-three blaOXA-48K. pneumoniae and two E. coli isolates were analyzed. The annual incidence of blaOXA-48K. pneumoniae isolates from 2012 to 2015 were 0%, 1.1%, 2.4%, and 7.6%, respectively. Forty-three (95.5%) of 45 isolates were non-susceptible to broad-spectrum beta-lactams (ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam), Forty-two (93.3%) of the 45 isolates showed resistance against all tested carbapenems (imipenem, meropenem, doripenem, and ertapenem). Molecular characterization revealed that they co-produced at least one extended-spectrum beta-lactamases or AmpC beta-lactamases, with at least one outer membrane porin loss. Thirty-eight (88.3%) of the 43 K. pneumoniae isolates belonged to ST11. PFGE analysis of 43 K. pneumoniae isolates revealed dissemination of multiple clones. Six of the 12 tested K. pneumoniae representatives of different pulso-types belonged to IncA/C. Concomitant loss of porins and production of other beta-lactamases renders the blaOXA-48-producing isolates in Taiwan a high-level carbapenem resistance and broad resistance against many beta-lactam antibiotics. Following dissemination of multiple clones of blaOXA-48K pneumoniae ST 11, a trend of increased blaOXA-48 prevalence was noted.

Molecular Epidemiology and Mechanisms of High-Level Resistance to Meropenem and Imipenem in Pseudomonas aeruginosa.

PurposePseudomonas aeruginosa possesses a large number of resistance mechanisms to different antimicrobials with carbapenems being the most powerful in treating resistant P. aeruginosa. Hence, it is imperative to explore different mechanisms of carbapenems-resistance in P. aeruginosa to achieve successful treatment through the design of new drugs acting on this interaction to combat against antimicrobial resistance.Strains and MethodsA total of 634 P. aeruginosa clinical isolates were collected from various patient sources and their MIC levels were measured. Molecular evaluation of carbapenem resistance was assessed by investigating the presence of blaIMP1, blaIMP2, blaVIM1, blaVIM2, blaSPM and blaNDM genes and the gene expression of the following multi-drug efflux pump systems: MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM and its correlation with MIC. Isolates were typed by Random Amplified Polymorphic DNA (RAPD)-typing.ResultsCarbapenem resistance was detected in 32 (5%) isolates, which were all imipenem resistant (of which 29 were meropenem resistant). High-level resistance (≥64mg/mL) to imipenem was found in 27 (84.3%) isolates, and to meropenem in 28 (96.5%) isolates. The carbapenemase blaVIM-1 was found in 31 isolates, while blaNDM was detected in 4 isolates. None of the isolates possessed either bla-VIM-2, blaIMP-1, blaIMP-2 or blaSPM. The majority of the isolates displayed over-expression of MexCD-OprJ (75%) followed by MexXY-OprM efflux pump (62%), while MexAB-OprM and MexEF-OprN efflux pumps were overexpressed in 21.8% and 18.7% of the isolates, respectively, with no down-regulation of oprD in any of the isolates. A strong correlation was found between CDJ efflux pump expression and meropenem, imipenem resistance (r=0.532, 0.654, p<0.001, <0.001) respectively. Four major clusters were detected by RAPD-typing: group 1(10 isolates), group 3 (9 isolates), group 2 (8 isolates) while the fourth group (4) included 4 isolates (12.5% polymorphism).ConclusionHigh-level carbapenem resistance reported in this study was allied to multiple mechanisms including carbapenemase production and efflux-pump over-expression. Threatening cross-infection is possible inside the hospital and stringent infection control measures are crucial.

Multiple carbapenemase gene production by Acinetobacter baumannii isolates from burn patients in Iran

Objective: Acinetobacter baumannii is among the major Gram-negative nosocomial pathogens, and their antibiotic resistance has spread extensively; especially in burn settings. Methods: A total of 300 clinical isolates of A. baumannii were collected from burn patients hospitalized in burn settings. The isolates were examined for antimicrobial susceptibility testing by the disc diffusion method. The class D (bla(OXA-51)-like, bla(OXA-23)-like, bla(OXA-58)-like and bla(OXA-24)-like) and class B (encoded by bla(VIM) and bla(SIM)) carbapenemase genes were investigated by multiplex PCR. Results: A high level of carbapenem resistance was observed among isolates, but none of them were resistant to colistin. Among carbapenem-resistant A. baumannii, previous antibiotic consumption was significantly higher (significant risk factor for carbapenem-resistant A. baumannii acquisition) than other risk factors (P = 0.0123), whereas older age of patients was not significantly higher among other ranges in multivariate analysis by analysis of variance (ANOVA test). The bla(OXA-51-like) gene was the predominant gene, followed by bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), bla(SIM), bla(NDM) and bla(VIM) genes being 66, 35.33, 22, 14, 1.33, 1.33 and 13.33%, respectively. Furthermore, the co-existence of bla(OXA-51-like)/bla(OXA-23-like), bla(OXA-51-like)/bla(OXA-23-like)/bla(OXA-24-like) and bla(OXA-51-like)/bla(OXA-24-like) were 22.67, 12 and 11.33%, respectively. Conclusion: A high level of class D and class B carbapenemases among A. baumannii strains in the burn settings is a crisis in the eradication of infections caused by MDR, XDR and PDR strains. Therefore, the uncontrolled consumption of last-line antibiotics should be restricted and infection control strategies must be implemented accurately. Copyright (C) 2019 Wolters Kluwer Health, Inc. All rights reserved.

Identification of the novel class D β-lactamase OXA-679 involved in carbapenem resistance in Acinetobacter calcoaceticus.

The aim of this study was to characterize the Acinetobacter calcoaceticus clinical isolate AC_2117 with the novel carbapenem-hydrolysing class D β-lactamase (CHDL) OXA-679. Identification of the species and β-lactamases was verified by genome sequencing (PacBio) and phylogenetic analyses. Antibiotic susceptibility of AC_2117 and transformants harbouring cloned blaOXA-679 was evaluated using antibiotic gradient strips and microbroth dilution. OXA-679 was purified heterologously and kinetic parameters were determined using spectrometry or isothermal titration calorimetry. The impact of OXA-679 production during imipenem therapy was evaluated in the Galleria mellonella infection model. Sequencing of the complete genome of the clinical A. calcoaceticus isolate AC_2117 identified a novel CHDL, termed OXA-679. This enzyme shared sequence similarity of 71% to each of the families OXA-143 and OXA-24/40. Phylogenetic analyses revealed that OXA-679 represents a member of a new OXA family. Cloning and expression of blaOXA-679 as well as measurement of kinetic parameters revealed the effective hydrolysis of carbapenems which resulted in reduced susceptibility to carbapenems in Escherichia coli and A. calcoaceticus, and high-level carbapenem resistance in Acinetobacter baumannii. Infection of larvae of G. mellonella with a sublethal dose of blaOXA-679-expressing A. baumannii could not be cured by high-dose imipenem therapy, indicating carbapenem resistance in vivo. We identified blaOXA-679 in a clinical A. calcoaceticus isolate that represents a member of the new OXA-679 family and that conferred high-level carbapenem resistance in vitro and in vivo.

Emergence of carbapenem resistance in Bacteroides fragilis in China

The antimicrobial resistance crisis makes it critically important for laboratories to closely monitor trends and mechanisms of emerging antimicrobial resistance in clinical isolates. Bacteroides fragilis is an anaerobic pathogen that causes several serious infections and is increasingly resistant to antimicrobial agents. However, data from China regarding antimicrobial resistance in B. fragilis are limited. In this work, the mechanism underlying carbapenem resistance in 44 B. fragilis isolates collected from a Chinese hospital was investigated. Antimicrobial susceptibility testing for 13 antimicrobial agents was performed by the agar dilution method, and the contribution of efflux pumps to carbapenem resistance was analysed. Genetic relatedness of the isolates was determined by PFGE. Outer membrane porins were analysed in isolates with reduced carbapenem susceptibility. Potential carbapenemase-encoding genes were identified, and the location and environment of the cfiA gene was analysed. Among the 44 isolates, 18.2%, 29.5%, 22.7%, 100%, 100%, 29.5%, 15.9%, 81.8%, 88.6% and 47.7% were resistant to imipenem, meropenem, ertapenem, penicillin, ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, clindamycin, tetracycline and moxifloxacin, respectively. None of the isolates were resistant to metronidazole, cefoxitin or chloramphenicol. A chromosomally located gene (cfiA) encoding a metallo-β-lactamase was identified in 16 isolates (36.4%). A conserved insertion sequence of IS1187 or IS613 was upstream of cfiA in eight isolates with high-level carbapenem resistance. The insertion sequences were associated with increased carbapenem resistance in B. fragilis by upregulating the expression of cfiA as shown by RT-qPCR. This is the first study to describe a mechanism of carbapenem resistance in B. fragilis in mainland China.

Phenotypic and molecular characterisations of carbapenem-resistant Acinetobacter baumannii strains isolated in Madagascar

BackgroundThe present study aimed to perform a deep phenotypic and genotypic analysis of 15 clinical carbapenem-resistant Acinetobacter baumannii (CRAb) strains isolated in Madagascar between 2008 and 2016 from diverse sources.MethodsCRAb isolates collected from the Clinical Biology Centre of the Institut Pasteur of Madagascar, from the neonatal unit of Antananarivo military hospital, and from intensive care units of Mahajanga Androva and Antananarivo Joseph Ravoahangy Andrianavalona (HJRA) hospitals were subjected to susceptibility testing. Whole-genome sequencing allowed us to assess the presence of antibiotic-resistance determinants, insertion sequences, integrons, genomic islands and potential virulence factors in all strains. The structure of the carO porin gene and deduced protein (CarO) were also assessed in CRAb isolates.ResultsAll isolates were found to be multidrug-resistant strains. Antibiotic-resistance genes against six classes of antimicrobial agents were described. The four carbapenem-resistance genes: blaOXA-51 like, blaOXA-23, blaOXA-24, and blaOXA-58 genes were detected in 100, 53.3, 13.3, and 6.6% of the isolates, respectively. Additionally, an ISAba1 located upstream of blaOXA-23 and blaADC-like genes was observed in 53.3 and 66.7% of isolates, respectively. Further, Tn2006 and Tn2008 were found associated to the ISAba1-blaOXA-23 structure. An 8051-bp mobilizable plasmid harbouring the blaOXA-24 gene was isolated in two strains. In addition, 46.7% of isolates were positive for class 1 integrons. Overall, five sequences types (STs), with predominantly ST2, were detected. Several virulence genes were found in the CRAb isolates, among which two genes, epsA and ptk, responsible for the capsule-positive phenotype, were involved in A. baumannii pathogenesis.ConclusionsThis study revealed the presence of high-level carbapenem resistance in A. baumannii with the first description of OXA-24 and OXA-58 carbapenemases in Madagascar. This highlights the importance of better monitoring and controlling CRAb in Madagascan hospitals to avoid their spread.

An ancient family of mobile genomic islands introducing cephalosporinase and carbapenemase genes in Enterobacteriaceae

ABSTRACT The exchange of mobile genomic islands (MGIs) between microorganisms is often mediated by phages, which may provide benefits to the phage’s host. The present study started with the identification of Enterobacter cloacae, Klebsiella pneumoniae and Escherichia coli isolates with exceptional cephalosporin and carbapenem resistance phenotypes from patients in a neonatal ward. To identify possible molecular connections between these isolates and their β-lactam resistance phenotypes, the respective bacterial genome sequences were compared. This unveiled the existence of a family of ancient MGIs that were probably exchanged before the species E. cloacae, K. pneumoniae and E. coli emerged from their common ancestry. A representative MGI from E. cloacae was named MIR17-GI, because it harbors the novel β-lactamase gene variant bla MIR17. Importantly, our observations show that the MIR17-GI-like MGIs harbor genes associated with high-level resistance to cephalosporins. Among them, MIR17-GI stands out because MIR17 also displays carbapenemase activity. As shown by mass spectrometry, the MIR17 carbapenemase is among the most abundantly expressed proteins of the respective E. cloacae isolate. Further, we show that MIR17-GI-like islands are associated with integrated P4-like prophages. This implicates phages in the spread of cephalosporin and carbapenem resistance amongst Enterobacteriaceae. The discovery of an ancient family of MGIs, mediating the spread of cephalosporinase and carbapenemase genes, is of high clinical relevance, because high-level cephalosporin and carbapenem resistance have serious implications for the treatment of patients with enterobacteriaceal infections.

Carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa: characterization of carbapenemase genes and E-test evaluation of colistin-based combinations.

BackgroundCarbapenamase producing Acinetobacter baumannii and Pseudomonas aeruginosa are emerging worldwide limiting the use of carbapenems as effective and safe drugs.PurposeTo characterize different carbapenemase genes carried by carbapenem-resistant (CR) A. baumannii and P. aeruginosa isolates and to evaluate the in vitro effect of some colistin-based combinations by E-test method in Zagazig University Hospitals ICU isolates.MethodsCR A. baumannii and P. aeruginosa isolated from the surgical intensive care unit (ICU) were tested for carbapenemase genes by polymerase chain reaction and the effect of colistin/meropenem and colistin/tigecycline combinations was evaluated by E-test.ResultsGenes coding for OXA-23, NDM and GES were detected in 90, 66.7 and 50% of CR A. baumannii, respectively, while genes coding for VIM, GES, NDM and IMP were detected in 50, 40.9, 27.3 and 18.2% of CR P. aeruginosa, respectively. Colistin/tigecycline combination showed synergistic and additive effect in 20% and 60% of A. baumannii isolates, respectively, while colistin/meropenem combination showed synergistic and additive effect in 63.6% and 36.4% of P. aeruginosa, respectively.ConclusionCarbapenemase genes carriage accounts for high level carbapenem resistance in our isolates. Colistin/tigecycline and colistin/meropenem combinations can be considered for treatment of severe infections by CR A. baumannii and P. aeruginosa, respectively.

SUSCEPTIBILITY TO ANTIBIOTICS IN KLEBSIELLA PNEUMONIAE STRAINS ISOLATED IN A MULTIDISCIPLINARY MEDICAL CENTRE

Susceptibility to 16 antimicrobial agents in 421 Klebsiella pneumoniae strains, isolated in a multidisciplinary medical centre from patients with nosocomial infections in 2015, was tested. The majority of studied strains were resistant to antibiotics: ampicillin/clavulanic acid (91.4%), ticarcillin/clavlanic acid (81.9%), piperacillin/tazobactam (69.4%), fluorochinolones (83.6%), III and IV generation of cephalosporines (79.8%), gentamycin (72.9%); one third (34.2%) demonstrated resistance to amikacin. K. pneumoniae strains demonstrated high level of carbapenem resistance (53.0% — to ertapenem, 42.8% — to meropenem and 37.1% — to imipenem), associated resistance to at least 3 different classes of antibiotics — caphalosporins, aminoglycozides, fluorochinolones, that amounted to more than half of the strains (57.7%), including 44.2% of the strains, additionally resistant to carbapenems. The lowest level of resistance was found to fosfomycin (8.5%) and tigecycline (7.4%), resistant cultures showed intermediate resistance with MIC 2 μg/ml to the latter. High diversity of antimicrobial resistance spectra was found, with high level of multidrug resistant strains (87.2%). Resistance to carapenems in K. pneumoniae isolated in the multidisciplinary medical center was determined by either blaOXA-48 (59.3% of isolates, resistant to carbapenems) or blaNDM-1 (40.7%).