Engineering 3-Hydroxypropionic Acid Production from Glucose in Yarrowia lipolytica through Malonyl-CoA Pathway.

3-Hydroxypropionic acid (3-HP) is an important intermediate compound in the chemical industry. Green and environmentally friendly microbial synthesis methods are becoming increasingly popular in a range of industries. Compared to other chassis cells, Yarrowia lipolytica possesses advantages, such as high tolerance to organic acid and a sufficient precursor required to synthesize 3-HP. In this study, gene manipulations, including the overexpression of genes MCR-NCa, MCR-CCa, GAPNSm, ACC1 and ACSSeL641P and knocking out bypass genes MLS1 and CIT2, leading to the glyoxylate cycle, were performed to construct a recombinant strain. Based on this, the degradation pathway of 3-HP in Y. lipolytica was discovered, and relevant genes MMSDH and HPDH were knocked out. To our knowledge, this study is the first to produce 3-HP in Y. lipolytica. The yield of 3-HP in recombinant strain Po1f-NC-14 in shake flask fermentation reached 1.128 g·L-1, and the yield in fed-batch fermentation reached 16.23 g·L-1. These results are highly competitive compared to other yeast chassis cells. This study creates the foundation for the production of 3-HP in Y. lipolytica and also provides a reference for further research in the future.

Non-negligible greenhouse gas emissions from non-sewered sanitation systems: A meta-analysis.

Current methods for estimating sanitation emissions underestimate the significance of methane emissions from non-sewered sanitation systems (NSSS), which are prevalent in many countries. NSSS play a vital role in the safe management of fecal sludge, accounting for approximately half of all existing sanitation provisions. We analyzed the distribution of global NSSS and used IPCC accounting methods to estimate the total methane emissions profiles from these systems. Then, we examined the literature to establish the level of uncertainty associated with this accounting estimate. The global methane emissions from NSSS in 2020 was estimated to as 377 (22-1003) Mt CO2e/year or 4.7% (0.3%-12.5%) of global anthropogenic methane emissions, which are comparable to the greenhouse gas (GHG) emissions from wastewater treatment plants. NSSS is the major option for open defecation and is expected to increase by 55 Mt CO2e/year after complete open defecation free. It is time to acknowledge the GHG emissions from the NSSS as a non-negligible source.

Climate Change Impacts on Urban Sanitation: A Systematic Review and Failure Mode Analysis.

Climate change will stress urban sanitation systems. Although urban sanitation uses various infrastructure types and service systems, current research appears skewed toward a small subset of cases. We conducted a systematic literature review to critically appraise the evidence for climate change impacts on all urban sanitation system types. We included road-based transport networks, an essential part of fecal sludge management systems. We combined the evidence on climate change impacts with the existing knowledge about modes of urban sanitation failures. We found a predominance of studies that assess climate impacts on centralized sewerage in high-income contexts. The implications of climate change for urban nonsewered and complex, fragmented, and (partially) decentralized sanitation systems remain under-researched. In addition, the understanding of the impacts of climate change on urban sanitation systems fails to take a comprehensive citywide perspective considering interdependencies with other sectors and combinations of climate effects. We conclude that the evidence for climate change impacts on urban sanitation systems is weak. To date, research neither adequately represents the variety of urban sanitation infrastructure and service systems nor reflects the operational and management challenges of already stressed systems.

Risk of COPD Exacerbations Associated with Statins versus Fibrates: A New User, Active Comparison, and High-Dimensional Propensity Score Matched Cohort Study.

BACKGROUND: Several observational studies have found that statins may materially decrease the risk of chronic obstructive pulmonary disease (COPD) exacerbations. However, most of these studies used a prevalent user, non-user comparison approach, which may lead to overestimation of the clinical benefits of statins. We aimed to explore the risk of COPD exacerbations associated with statins with a new user, active comparison approach to address potential methodological concerns. We selected fibrates, another class of lipid-lowering agents, as the reference group because no evidence suggests that fibrates have an effect on COPD exacerbations. METHODS: We identified patients with COPD who initiated statins or fibrates from a nationwide Taiwanese database. Patients were followed from cohort entry to the earliest of the following: hospitalization for COPD exacerbations, death, end of the data, or 180 days after cohort entry. Stratified Cox regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of COPD exacerbations comparing statins with fibrates after variable-ratio propensity score (PS) matching and high-dimensional PS (hd-PS) matching, respectively. RESULTS: We identified a total of 134,909 eligible patients (110,726 initiated statins; 24,183 initiated fibrates); 1979 experienced COPD exacerbations during follow-up. The HRs were 1.10 (95% CI, 0.96 to 1.26) after PS matching and 1.08 (95% CI, 0.94 to 1.24) after hd-PS matching. The results did not differ materially by type of statins and patient characteristic and did not change with longer follow-up durations. CONCLUSION: This large-scale, population-based cohort study did not show that use of statins was associated with a reduced risk of acute exacerbations in patients with COPD using state-of-the-art pharmacoepidemiologic approaches. The findings emphasize the importance of applying appropriate methodology in exploring statin effectiveness in real-world settings.

Comparative genomic analyses of IncpA1763-KPC plasmids.

Multi-replicon plasmids harboring the IncpA1763-KPC replicon together with other replicons are being increasingly reported among Enterobacteriaceae species. However, plasmids with single IncpA1763-KPC replicons are poorly studied as a different incompatibility (Inc) group, despite their rise in appearance in some strains. IncpA1763-KPC plasmids, pA1763-KPC, and p427113-2, from two clinical Klebsiella pneumoniae isolates were fully sequenced by high-throughput genome sequencing. Linear structural comparisons of IncpA1763-KPC backbone region were made between these two plasmids and six arbitrarily selected representative IncpA1763-KPC plasmids sequenced previously. A further detailed genomic comparison was carried out between plasmids pA1763-KPC, p427113-2, and pFB2.2, which show high homology across the backbone sequence to one another. Among all sequenced IncpA1763-KPC plasmids considered in this study, plasmids pA1763-KPC and p427113-2 showed the most complete IncpA1763-KPC backbones. These were composed of the IncpA1763-KPC replicon (repAIncpA1763-KPC and its iterons), the 5.6-kb IncpA1763-KPC -type maintenance region, the 27.7-kb IncFIIK -type maintenance region, and the 36.6-kb IncFIIK -type conjugal transfer regions. Compared with pA1763-KPC or p427113-2, the backbone regions of the other analyzed IncpA1763-KPC plasmids had gradually undergone different deletions or truncations, but shared small and core IncpA1763-KPC backbones including the IncpA1763-KPC replicon, IncpA1763-KPC -type maintenance region, and residual IncFIIK -type maintenance region. Accessory modules integrated into IncpA1763-KPC backbones included the multidrug-resistant module blaKPC-2 region in pA1763-KPC, the metal-resistance modules ars region together with ncr region in pFB2.2 and sil in pKPN-9a0d, the ISKpn14-to-IS26 region in p427113-2, and other non-resistance region in the respective plasmids. This detailed comparative genomics analysis of IncpA1763-KPC plasmids provides a deep insight into their diversification and evolution.

Comparative Performance of Comorbidity Measures in Predicting Health Outcomes in Patients with Chronic Obstructive Pulmonary Disease.

Purpose: Multiple studies have suggested that comorbidities pose negative impacts on the survival of patients with chronic obstructive pulmonary disease (COPD); few have applied comorbidity measures driven from health insurance claims databases to predict various health outcomes. We aimed to examine the performance of commonly used comorbidity measures based on diagnosis and pharmacy dispensing claims information in predicting future death and hospitalization in COPD patients. Methods: We identified COPD patients in a population-based Taiwanese database. We built logistic regression models with age, sex, and baseline comorbidities measured by either diagnosis or pharmacy claims information as predictors of subsequent-year death or hospitalization in a random 50% sample and validated the discrimination in the other 50%. The diagnosis-based comorbidity measures included the Charlson Index and the Elixhauser comorbidity measure; the pharmacy-based comorbidity measures included the updated Chronic Disease Score (CDS) and the Pharmacy-Based Comorbidity Index (PBDI). Results: We identified 428,251 eligible patients. For overall death, the Elixhauser comorbidity measure showed the best predictive performance (c-statistic=0.832), followed by the PBDI (c-statistic=0.822), the Charlson Index (c-statistic=0.815), and the updated CDS (c-statistic=0.808). For overall hospitalization, the PBDI (c-statistics=0.730) and the Elixhauser comorbidity measure (c-statistics=0.724) outperformed the updated CDS (c-statistics=0.714) and the Charlson Index (c-statistics=0.710). For hospitalization due to cardiovascular, cerebrovascular, or respiratory diseases, the comorbidity models showed similar predictive ranks and demonstrated c-statistics higher than 0.75. However, none of the models could adequately predict hospitalization due to other reasons (c-statistics < 0.60). Conclusion: Our study comprehensively compared the predictive performance of comorbidity measures. The Elixhauser comorbidity measure and the PBDI are useful tools for describing comorbid conditions and predicting health outcomes in COPD patients.

Genetic Characterization of a bla VIM-24-Carrying IncP-7ß Plasmid p1160-VIM and a bla VIM-4-Harboring Integrative and Conjugative Element Tn6413 From Clinical Pseudomonas aeruginosa.

This study presents three novel integrons In1394, In1395, and In1443, three novel unit transposons Tn6392, Tn6393, and Tn6403, one novel conjugative element (ICE) Tn6413, and the first sequenced IncP-7 resistance plasmid p1160-VIM from clinical Pseudomonas aeruginosa. Detailed sequence comparison of p1160-VIM (carrying Tn6392 and Tn6393) and Tn6413 (carrying Tn6403) with related elements were performed. Tn6392, Tn6393, and Tn6403 were generated from integration of In1394 (carrying bla VIM-24), In1395 and In1443 (carrying bla VIM-4) into prototype Tn3-family unit transposons Tn5563, Tn1403, and Tn6346, respectively. To the best of our knowledge, this is the first report of a bla VIM-24-carrying P. aeruginosa isolate.

Replicon-Based Typing of IncI-Complex Plasmids, and Comparative Genomics Analysis of IncIγ/K1 Plasmids.

IncI-complex plasmids can be divided into seven subgroups IncI1, IncI2, IncIγ, IncB/O, IncK1, IncK2, and IncZ. In this study, a replicon-based scheme was proposed for typing IncI-complex plasmids into four separately clustering subgroups IncI2, IncI1/B/O, IncIγ/K1 and IncK2/Z, the last three of which were combined from IncI1 and IncB/O, IncIγ and IncK1, and IncK2 and IncZ, respectively. Four IncIγ/K1 plasmids p205880-NR2, p14E509-CTXM, p11011-CTXM and p61806-CTXM were fully sequenced and compared with IncIγ/K1 reference pCT, IncI2 reference R721, IncI1/B/O reference R64 and IncK2/Z reference pO26-CRL-125. These plasmids shared conserved gene organization in the replication and conjugal transfer regions, but displaying considerable sequence diversity among different subgroups. Remarkable modular differences were observed in the maintenance and transfer leading regions. p205880-NR2 contained no resistance genes or accessory modules, while the other seven plasmids acquired one or more accessory modules, which harbored mobile elements [including unit transposons, insertion sequence (IS)-based transposition units and individual IS elements] and associated resistance markers (especially including those involved in resistance to ß-lactams, aminoglycosides, tetracyclins, phenicols, streptomycins, trimethoprims, sulphonamides, tunicamycins and erythromycins). Data presented here provided a deeper insight into diversification and evolution of IncI-complex plasmids.

Comparative analysis of bla KPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids from Klebsiella pneumoniae CG258 strains disseminated among multiple Chinese hospitals.

BACKGROUND: We recently reported the complete sequence of a blaKPC-2- and rmtB-carrying IncFII-family plasmid p675920-1 with the pKPC-LK30/pHN7A8 hybrid structure. Comparative genomics of additional sequenced plasmids with similar hybrid structures and their prevalence in bla KPC-carrying Klebsiella pneumoniae strains from China were investigated in this follow-up study. METHODS: A total of 51 bla KPC-carrying K. pneumoniae strains were isolated from 2012 to 2016 from five Chinese hospitals and genotyped by multilocus sequence typing. The bla KPC-carrying plasmids from four representative strains were sequenced and compared with p675920-1 and pCT-KPC. Plasmid transfer, carbapenemase activity determination, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids. The prevalence of pCT-KPC-like plasmids in these bla KPC-carrying K. pneumoniae strains was screened by PCR. RESULT: The six KPC-encoding plasmids p1068-KPC, p20049-KPC, p12139-KPC and p64917-KPC (sequenced in this study) and p675920-1 and pCT-KPC slightly differed from one another due to deletion and acquisition of various backbone and accessory regions. Two major accessory resistance regions, which included the blaKPC-2 region harboring blaKPC-2 (carbapenem resistance) and blaSHV-12 (ß-lactam resistance), and the MDR region carrying rmtB (aminoglycoside resistance), fosA3 (fosfomycin resistance), bla TEM-1B (ß-lactam resistance) and bla CTX-M-65 (ß-lactam resistance), were found in each of these six plasmids and exhibited several parallel evolution routes. The pCT-KPC-like plasmids were present in all the 51 K. pneumoniae isolates, all of which belonged to CG258. CONCLUSION: There was clonal dissemination of K. pneumoniae CG258 strains, harboring bla KPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids, among multiple Chinese hospitals.

Plasmid and chromosomal integration of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.

Objectives: To provide detailed genetic characterization of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp. Methods: P. aeruginosa 60512, K. pneumoniae 447, P. aeruginosa 12939 and Enterobacter sp. A1137 were subjected to genome sequencing. The complete nucleotide sequences of two plasmids (p60512-IMP from the 60512 isolate and p447-IMP from the 447 isolate) and two chromosomes (the 12939 and A1137 isolates) were determined, then a genomic comparison of p60512-IMP, p447-IMP and four novel blaIMP-carrying transposons (Tn6394, Tn6375, Tn6411 and Tn6397) with related sequences was performed. Transferability of the blaIMP gene and bacterial antimicrobial susceptibility were tested. Results: Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP, respectively, while Tn6411 and Tn6397 were integrated into the 12939 and A1137 chromosomes, respectively. Tn6394 was an ISPa17-based transposition unit that harboured the integron In992 (carrying blaIMP-1). In73 (carrying blaIMP-8), In73 and In992, together with the ISEcp1:IS1R-blaCTX-M-14-IS903D unit, the macAB-tolC region and the truncated aacC2-tmrB region, respectively, were integrated into the prototype transposons Tn1722, Tn1696 and Tn7, respectively, generating the Tn3-family unit transposons, Tn6375 and Tn6378, and the Tn7-family unit transposon Tn6411, respectively. Tn6397 was a large integrative and conjugative element carrying Tn6378. Conclusions: Complex events of transposition and homologous recombination have occurred during the original formation and further plasmid and chromosomal integration of these four transposons, promoting accumulation and spread of antimicrobial resistance genes.

Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant Citrobacter freundii isolate.

BACKGROUND: Plasmid-mediated multi-drug resistance (MDR) has been widely found in Citro-bacter freundii. C. freundii P10159 was isolated from a human case of postoperative urinary tract infection in a Chinese teaching hospital. METHODS: The complete nucleotide sequences of five resistance plasmids pP10159-1, pP10159-2, pP10159-3, pP10159-4 and pP10159-5 from C. freundii P10159 were determined through high-throughput genome sequencing, and then compared with related plasmids sequences. Plasmid transfer, CarbaNP test of carbapenemase activity, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids. RESULTS: pP10159-1 carrying blaNDM-1and pP10159-2 harboring blaIMP-4 plus qnrS1 were almost identical to IncX3 plasmid pNDM-HN380 and IncN1 plasmid pP378-IMP, respectively. The blaKPC-2-carrying plasmids pP10159-3, pHS062105-3 and pECN49-KPC were highly similar to each other, and constituted a novel group of plasmids belonging to an unknown incomparability group. The MDR plasmids pP10159-4 and pP10159-5 had the backbones highly similar to IncHI4 plasmid pNDM-CIT and type 2 IncC plasmid pR55, respectively, but their accessory resistance regions differed from pNDM-CIT and pR55, respectively. The five plasmids from the P10159 isolate contained a total of 24 different genes or gene loci, which contributed to resistance to 13 distinct antibiotic molecules or toxic compounds. CONCLUSION: This is the first report of co-occurrence of five different resistance plasmids, with determination of their complete sequences. Data presented here provide a deeper insight into co-selection and maintenance of multiple plasmids and an extremely large number of resistance genes in a single bacterial isolate.

Dissemination of KPC-2-Encoding IncX6 Plasmids Among Multiple Enterobacteriaceae Species in a Single Chinese Hospital.

Forty-five KPC-producing Enterobacteriaceae strains were isolated from multiple departments in a Chinese public hospital from 2014 to 2015. Genome sequencing of four representative strains, namely Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20, indicated the presence of blaKPC-2-carrying IncX6 plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC in the four strains, respectively. These plasmids were genetically closely related to one another and to the only previously sequenced IncX6 plasmid, pKPC3_SZ. Each of the plasmids carried a single accessory module containing the blaKPC-2/3-carrying ΔTn6296 derivatives. The ΔTn6292 element from pGN26-KPC also contained qnrS, which was absent from all other plasmids. Overall, pKPC3_SZ-like blaKPC-carrying IncX6 plasmids were detected by PCR in 44.4% of the KPC-producing isolates, which included K. aerogenes, P. mirabilis, S. marcescens, M. morganii, Escherichia coli, and Klebsiella pneumoniae, and were obtained from six different departments of the hospital. Data presented herein provided insights into the genomic diversity and evolution of IncX6 plasmids, as well as the dissemination and epidemiology of blaKPC-carrying IncX6 plasmids among Enterobacteriaceae in a hospital setting.

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids.

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1α, IncX1ß and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (ß-lactam resistance), blaKPC-2 (carbapenem resistance) and ΔblaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids.

Co-occurrence of 3 different resistance plasmids in a multi-drug resistant Cronobacter sakazakii isolate causing neonatal infections.

Cronobacter sakazakii 505108 was isolated from a sputum specimen of a neonate with severe pneumonia. C. sakazakii 505108 co-harbors 3 resistance plasmids of the IncHI2, IncX3, and IncFIB incomparability groups, respectively. These 3 plasmids have acquired several accessory modules, which carry an extremely large number of resistance genes, especially including those involved in resistance to carbapenems, aminoglycoside, tetracyclines, and phenicols and sulphonamide/trimethoprim. These plasmid-borne antibiotic resistance genes were associated with insertion sequences, integrons, and transposons, indicating that the assembly and mobilization of the corresponding accessory modules with complex chimera structures are facilitated by transposition and/or homologous recombination. This is the first report of fully sequence plasmids in clinical Cronobacter, which provides a deeper insight into plasmid-mediated multi-drug resistance in Cronobacter from hospital settings.

Comparative genomics of type 1 IncC plasmids from China.

AIM: This study dealt with genomic characterization of type 1 IncC resistance plasmids, capable of spreading across taxonomic borders, from China. MATERIALS & METHODS: p112298-tetA was sequenced and compared with type 1 IncC reference plasmid pR148 and two available sequenced type 1 IncC plasmids pHS36-NDM and pVAS3-1 from China. RESULTS: These plasmids contained one or more exogenous resistance islands, which included the ARI-A islands, the ARI-B islands, the ISEcp1-blaCMY units and the bla KPC-2 region and were inserted at various sites in the IncC backbone and thus represented three distinct lineages. CONCLUSION: Complex rearrangement and homologous recombination events have occurred during evolution of p112298-tetA, making it significantly differ modularly from the other three plasmids with respect to both plasmid backbone and exogenous resistance regions.

Structural genomics of pNDM-BTR harboring In191 and Tn6360, and other bla NDM-carrying IncN1 plasmids.

AIM: To characterize a conjugative bla NDM-1-carrying plasmid pNDM-BTR from a clinical Escherichia coli isolate. MATERIALS & METHODS: The complete nucleotide sequence of pNDM-BTR was determined using next-generation sequencing technology. Comparative genomic analysis of bla NDM-carrying IncN1 plasmids, including pNDM-BTR, was performed, and the antimicrobial resistance phenotypes were determined. RESULTS: pNDM-BTR contained three accessory modules, namely IS26, a novel Tn3-family transposon Tn6360 and the dfrA14 region composed of In191, ecoRII-ecoRIImet and ΔIS1X2. The relatively small IncN1 backbones could integrate massive accessory modules, most of which were integrated at two 'hotspots'. These IncN1 plasmids contained distinct profiles of accessory modules, which included those carrying various resistance genes. CONCLUSION: This study provides a deeper insight into horizontal transfer of resistance genes among IncN1 plasmids.

pSY153-MDR, a p12969-DIM-related mega plasmid carrying blaIMP-45 and armA, from clinical Pseudomonas putida.

This work characterized mega plasmid pSY153-MDR, carrying blaIMP-45 and armA, from a multidrug-resistant (MDR) Pseudomonas putida isolate from the urine of a cerebral infarction patient in China. The backbone of pSY153-MDR was closely related to Pseudomonas plasmids p12969-DIM, pOZ176, pBM413, pTTS12, and pRBL16, and could not be assigned to any of the known incompatibility groups. The accessory modules of pSY153-MDR were composed of 10 individual insertion sequence elements and two different MDR regions, and differed dramatically from the above plasmids. Fifteen non-redundant resistance markers were identified to be involved in resistance to at least eight distinct classes of antibiotics. All of these resistance genes were associated with mobile elements, and were embedded within the two MDR regions. blaIMP-45 and armA coexisted in a Tn1403-Tn1548 region, which was generated from homologous recombination of Tn1403- and Tn1548-like transposons. The second copy of armA was a component of the ISCR28-armA-∆ISCR28 structure, representing a novel armA vehicle. This vehicle was located within In48, which was related to In363 and In1058. Data presented here provide a deeper insight into the evolutionary history of SY153, especially in regard to how it became extensively drug-resistant.

p1220-CTXM, a pKP048-related IncFIIK plasmid carrying bla CTX-M-14 and qnrB4.

AIM: This study aimed to characterize plasmid-mediated antimicrobial resistance in clinical Klebsiella pneumoniae 1220 carrying bla CTX-M-14 and qnrB4. MATERIALS & METHODS: Plasmid p1220-CTXM was transformed from the 1220 isolate into Escherichia coli through conjugal transfer and then fully sequenced. Antimicrobial susceptibility was determined by VITEK. RESULTS: p1220-CTXM was an IncFIIK plasmid genetically closely related to pKP048 and carried resistance markers including bla CTX-M-14, bla DHA-1, qnrB4, sul1 and qacEΔ1, all of which were harbored in a 35.7-kb multidrug-resistant region. bla CTX-M-14 was located in a truncated ISEcp1-bla CTX-M-14-orf477 transposition unit, and qnrB4 and bla DHA-1 were in a truncated qnrB4-bla DHA-1 region. CONCLUSION: This study provided the insight into the co-occurrence of bla CTX-M-14 and qnrB4 and the evolution of pKP048-related IncFIIK plasmids.

Sequencing and comparative genomics analysis of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA.

Incompatibility group IncHI plasmids are important vectors of antibiotic resistance in Enterobacteriaceae. In this study, a scheme for typing IncHI into five separately clustering subgroups, including previously designated IncHI1-3 as well as IncHI4-5, was proposed based on sequenced IncHI plasmids. The complete nucleotide sequences of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA from clinical Enterobacter cloacae, Citrobacter freundii and Raoultella ornithinolytica isolates, respectively, were determined and were compared with IncHI2 and IncHI5 reference plasmids. Considerable genetic conservation was observed within the backbone sequences of each of the IncHI2 and IncHI5 subgroups, but the backbone sequences of the two subgroups were dramatically different from each other. However, the conjugal transfer regions tra1 and tra2 as well as the tellurium resistance gene cluster ter were present in all five plasmids. A number of accessory regions associated with integrons, transposons and insertion sequence-based mobile elements have been inserted at various sites of the plasmid backbones, among which were several large regions harbouring genes conferring resistance to multiple classes of antibiotics. Data generated from this study provide us with a deeper understanding of the diversification of IncHI-type resistance plasmids.