Epidemic Plasmid Carrying bla CTX-M-15 in Klebsiella penumoniae in China

Objective

To investigate the local epidemiology of Klebsiella penumoniae carrying bla _CTX-M-15 in southern China and to characterize the genetic environment of bla _CTX-M-15.

Methods

PCR and DNA sequencing were used to detect and characterize the genetic contexts of bla _CTX-M-15. The clonal relatedness of isolates carrying bla _CTX-M-15 was determined by pulse-field gel electrophoresis. Conjugative plasmids carrying bla _CTX-M-15 were obtained by mating and were further subject to restriction analysis and replicon typing.

Results

A total of 47CTX-M-15 ESBL-producing isolates of K. pneumoniae were collected from nine hospitals in China from October 2007 to October 2008. Isolates were clustered into various clonal groups. The local spread of bla _CTX-M-15 was mainly mediated by one major conjugative plasmid as determined by S1-PFGE and restriction analysis. A 90-kb plasmid belonging to incompatible group FII was the major carrier of bla _CTX-M-15 in K. pneumoniae. Except bla _TEM-1, the resistance genes such as bla _SHV, bla _DHA-1, bla _OXA-1, qnrB, qnrS, aac(3)-II, and aac(6′)-Ib were not found in the plasmid. In the comparing of conjugative gene sequence, it is 100% identical with the plasmid pKF3–94, which was found in K. pneumonia from Zhejiang province of china previously.

Conclusions

bla _CTX-M-15 was prevalent in K. pneumonia of southern China. The dissemination of bla _CTX-M-15 appeared to be due to the horizontal transfer of a 90-kb epidemic plasmid.     

Molecular Characterization of an Atypical IncX3 Plasmid pKPC-NY79 Carrying bla KPC-2 in a Klebsiella pneumoniae

The IncX family of plasmids has recently been expanded to include at least four subtypes, IncX1–IncX4. The revised classification provides an opportunity for improving our understanding of the sequence diversity of the IncX plasmids and the resistance genes they carried. We described the complete nucleotide sequence of a novel IncX3 plasmid, pKPC-NY79 (42,447 bp) from a sequence-type 258 Klebsiella pneumoniae strain that was isolated from a patient who was hospitalized in New York, United States. In pKPC-NY79, the plasmid scaffold and genetic load region were highly similar to homologous regions in pIncX-SHV (IncX3, JN247852) and the bla _KPC carrying pKpQIL (IncFII_k, GU595196), respectively, indicating that it has possibly arisen through recombination of plasmids. The bla _KPC-2 gene, as part of a transposon Tn4401a, was found within the genetic load region. The backbone of pKPC-NY79 differs from pIncX-SHV by a deletion involving the gene tandem hns–topB (encoding H-NS protein and topoisomerase III, respectively) and a putative ATPase gene. Unexpectedly, the impact of the hns–topB deletion on host fitness and plasmid stability was found to be small. In conclusion, the findings contribute to a better understanding of the plasmid platforms carrying bla _KPC and of variations in the backbone of the IncX3 plasmids.     

Genomic analysis of a Kpi (pilus system)-positive and CTX-M-15-producing Klebsiella pneumoniae belonging to the high-risk clone ST15 isolated from an impacted river in Brazil

Convergence of resistance and virulence in Klebsiella pneumoniae is a critical public health issue worldwide. A multidrug-resistant CTX-M-15-producing K. pneumoniae (TIES-4900 strain) was isolated from a highly impacted urban river, in Brazil. The genome was sequenced by MiSeq Illumina platform and de novo assembled using Unicycler. In silico prediction was accomplished by bioinformatics tools. The size of the genome is 5.4 Mb with 5145 protein-coding genes. TIES-4900 strain belonged to the sequence type ST15, yersiniabactin sequence type YbST10, ICEKp4, KL24 (wzi-24) and O1v1 locus. Phylogenomics confirmed genomic relatedness with ST15 clones from human and animal hosts. Convergence of broad resistome (antibiotics, heavy-metals and biocides) and virulome, including the Kpi pilus system involved in host-pathogen interaction and persistence of ST15 clone to hospital environments, were predicted. Virulent behavior was confirmed in the Galleria mellonella infection model. This study may give genomic insights on the spread of critical-priority WHO pathogens beyond hospital settings.     

Occurrence of Yeasts in Cloacae of Migratory Birds

Several species of yeast have been reported as pathogens in humans based on increases in immunodeficiency syndromes and as a result of immunosuppressant chemotherapy in cancer treatment. Domestic and wild birds are known to act as carriers of human pathogenic fungi. To gain additional information on the yeasts present in the cloacae of some species of migratory birds, 421 wild birds (24.39% out of 1726 birds caught in Romania, Hungary and Bulgaria) were sampled with the permission of the local judicial authority. The state of conservation of the birds (i.e. post-mortem alterations, colour of the mucosae etc.), along with their age and sex were determined. Samples were collected directly from the cloacae and cultured, and colonies were identified in each positive sample. Yeasts were isolated from 15.7% of the animals sampled, with the highest percentage found in coots (Fulica atra −58.8%) and the lowest in quails (Coturnix coturnix −1.7%). A total of 131 isolates belonging to 15 species of yeast were identified. Rhodotorula rubra was the yeast with the highest number of isolates (28.2%), followed by Cryptococcus albidus (18.4%), Candida albicans (9.2%), Trichosporon cutaneum (8.4%), Candida guilliermondii (6.1%), Candida tropicalis (6.1%) and other species. The present study represents the first survey on the occurrence of yeasts in the cloacae of migratory birds. The prevalence and species of yeasts isolated is discussed on the basis of the ecology, diet, and habitat of the birds.     

Characteristics of Klebsiella pneumoniae harboring QnrB32, Aac(6')-Ib-cr, GyrA and CTX-M-22 genes

Quinolone resistance in members of the Enterobacteriaceae family is mostly due to mutations in the quinolone resistance-determining regions of topoisomerase genes. CTX-M-22 is a member of the CTX-M family which can reduce extended-spectrum β-lactamase (ESBL) production and modulate antibiotic resistance, resulting in low ceftazidime minimum inhibitory concentrations (MICs). There are four different genes in Klebsiella pneumoniae (KP4707) including qnrB32 (novel qnr allele gene, HQ704413), aac(6')-Ib-cr (novel aac(6')-Ib allele gene, HQ680690), gyrA (novel gyrA allele gene, HQ680691) and CTX-M-22 gene. Five point amino acid mutations Arn(N)27 → Leu(L), Val(V)129 → Ala(A), Iie(I)142 → Met(M), Gly(G)188 → Arg(R), Val(V)212 → Iie(I) were observed in the qnr32 gene when compared to qnrB1. Of all qnrB alleles, a novel variant of the qnrB32 gene, with qnrB31, had the highest amino acid homology. Three point amino acid mutations including Trp(W)105 → Arg(R), Asp(D)182 → Tyr(Y) and Val(V)201 → Asp(D) were observed in aac(6')-Ib-cr gene, when compared to GenBank number AF479774. New variants of qnr32, aac(6')-Ib-cr, gyrA and CTX-M-22 or other genotype determinants continuously appear in different genomic sites and also outside the Enterobacteriaceae family.     

pIMP-PH114 Carrying bla IMP-4 in a Klebsiella pneumoniae Strain is Closely Related to Other Multidrug-Resistant IncA/C2 Plasmids

The IncA/C plasmids are broad host-range vehicles which have been associated with wide dissemination of CMY-2 among Enterobacteriaceae of human and animal origins. Acquired metallo-β-lactamases (MBLs) such as the IMP-type enzymes are increasingly reported in multidrug-resistant Gram-negative bacteria worldwide, particularly in Enterobacteriaceae. We described the complete sequence of the first IMP-4-encoding IncA/C₂ plasmid, pIMP-PH114 (151,885 bp), from a sequence type 1 Klebsiella pneumoniae strain that was recovered from a patient who was hospitalized in the Philippines. pIMP-PH114 consists of a backbone from the IncA/C₂ plasmids, with the insertion of a novel Tn21-like class 1 integron composite structure (containing the cassette array bla _IMP-4-qacG-aacA4-catB3, followed by a class C β-lactamase bla _DHA-1 and the mercury resistance operon, merRTPCADE) and a sul2-floR encoding region. Phylogenetic analysis of the IncA/C repA sequences showed that pIMP-PH114 formed a subgroup with other IncA/C plasmids involved in the international spread of CMY-2, TEM-24 and NDM-1. Identical bla _IMP-4 arrays have been described among different Enterobacteriaceae and Acinetobacter spp. in China, Singapore and Australia but the genetic context is different. The broad host range of IncA/C plasmids may have facilitated dissemination of the bla _IMP-4 arrays among different diverse groups of bacteria.     

Molecular epidemiology of antibiotic-resistant genes and potent inhibitors against TEM,CTX-M-14, CTX-M-15, and SHV-1 proteins of Escherichia coli in district Peshawar,Pakistan

The Extended Spectrum Beta-Lactamases (ESBLs) producing bacteria is an issue of concern for clinicians resulting in minimize the treatment options. To overcome resistance mechanisms, novel inhibitors with good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties must inhibit the ESBLs resistant genes. The current study aimed to identify the antibiotic resistance genes of ESBLs producing E. coli and a single inhibitor was designed to inhibit all the resistant proteins. The results showed that 42.9% ESBL producers had CTX-M (69.9%), TEM (63.4%), SHV (34.5%) and CTX-M-14 (17.5%) genes. The ESBLs producing isolates were resistant to cephalosporins, quinolones, and sulfonamide with Minimum Inhibitory Concentration (MICs) ranging from 64 to >256 μg/ml. To design multi inhibitory ligands, RECAP synthesis was used for the de-novo discovery of 1000 inhibitors database. Protein crystal structures were retrieved from Protein Data Base (PDB). Lipinski’s rules of five were applied to the novel inhibitors database to improve the ADMET properties. The novel inhibitors database was selected for docking simulations. Placement of the ligand was used by the London dG algorithm implemented in Molecular Operating Environment (MOE), while GBVI/WSA dG algorithm was used for final refinement. Based on docking score, visual inspection of ligands interaction with key residues, binding affinity, and binding energy of ligands with proteins, ten compounds were selected for ESBLs proteins with best ADMET properties, binding energy, and binding affinity the reported ones. These hits compounds have unique scaffolds and are predicted to be a starting point for developing potent inhibitors against antibiotic-resistant proteins.     

沈阳周边重要生态保护地春季迁徙鸟类多样性调查

2014年3月21日—5月9日,采用样线法与定点观察法对沈阳市周边26个重要生态保护地春季迁徙期鸟类多样性进行了调查。共记录鸟类16目40科94种。其中,国家Ⅰ级重点保护鸟类2种;国家Ⅱ级重点保护鸟类4种。居留型组成以夏候鸟和旅鸟为主,共占记录鸟类物种总数的77.7%。区系组成以古北界种为主,占74.5%。生态型中鸣禽最多,占36.2%。不同调查样地的鸟类组成与多样性指数存在较大差异,水库湿地鸟类数量最多;湿地公园物种丰富度最高;森林生态系统鸟类多样性较高;沙地生境鸟类多样性较低。调查发现部分生态保护地存在人为干扰程度较大、生态破坏严重、生境类型高度单一等生态问题。针对相应的生态保护地应进一步加强生态环境治理,为鸟类生存提供良好的栖息环境。     

聚羟基丁酸路径在克雷伯氏菌中的构建

以生物柴油的副产物甘油生产高附加值的1,3-丙二醇,现已成为提升生物柴油产业链经济性的重要途径,而中间代谢产物3-羟基丙醛积累造成细胞死亡,发酵异常终止是生物法生产1,3-丙二醇过程中的关键问题。不同于传统的降低3-羟基丙醛积累的思路,本文从增强克雷伯氏菌对3-羟基丙醛的抗逆性出发,改善克雷伯氏菌1,3-丙二醇的生产性能,首次将聚羟基丁酸路径引入克雷伯氏菌中,构建了新型基因工程菌,并对其1,3-丙二醇发酵性能及聚羟基丁酸代谢进行了初步的研究。经IPTG诱导,工程菌中检测到聚羟基丁酸,其含量随IPTG浓度增加而增大。优化的IPTG浓度为0.5 mmol/L。初始甘油50 g/L时,野生菌可正常发酵生产1,3-丙二醇,1,3-丙二醇浓度达到22.1 g/L,其质量得率为46.4%。当初始甘油达到70 g/L时,由于高浓度3-HPA积累,野生菌发酵终止,而工程菌可正常发酵生产1,3-丙二醇,PDO产量可达31.3 g/L,其质量得率为43.9%。同时检测到聚羟基丁酸积累。研究结果有助于加深对克雷伯氏菌1,3-丙二醇代谢机理的认识,为克雷伯氏菌的进一步优化提供了新的思路。     

Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital

Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla _CTX-M-15 (12 isolates), bla _CTX-M-14a (one isolate), and bla _CTX-M-14b (one isolate). The bla _OXA-1 gene was detected in 13 bla _CTX-M-producing strains and a bla _TEM-1 gene in 6 of them. The ISEcp1 sequence was found upstream of bla _CTX-M genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17–aadA5 being the most common. One of the strains (bla _CTX-M-14a-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6′)-Ib and cmlA1 genes and it was linked to the bla _CTX-M-14a gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.     

候鸟迁徙过程中"雄性早现"的六种假说及未来研究热点

"雄性早现"(protandry)是指雄性相对于雌性更早进入繁殖状态或更早到达繁殖地的现象.该文针对候鸟的雄性比雌性在春季时更早到达繁殖地这一现象,介绍了雄性早现的6种假说,即等级优势(rank advantage)假说、敏感性(susceptibility)假说、限制性(constraint)假说、交配机会(mate opportunity)假说、等待代价(waiting cost)假说和配偶选择(mate choice)假说,并通过近年来的研究证据阐述了上述假说对解释候鸟雄性早现的适用性.此外,对鸟类雄性早现未来研究中可能的热点问题做了展望.     

The efficacy of Klebsiella pneumoniae bacteriophage in the therapy of experimental Klebsiella infection

The effectiveness of specific phage therapy was studied on Klebsiella experimental sepsis in noninbred white mice, caused by the intraperitoneal injection of K. pneumoniae highly virulent strain K2 5055 into the animals. For treatment, Klebsiella polyvalent bacteriophage administered on day 2 after the infection of the animals with Klebsiella was used. The study revealed that bacteriophage could be detected in the blood and internal organs of the animals within 24 hours irrespective of the route of its administration: intraperitoneal, intravenous or intranasal. The bacteriophage preparation, introduced intraperitoneally, was shown to be effective in the treatment of generalized Klebsiella infection. One daily intraperitoneal injection of Klebsiella bacteriophage for 15-20 days proved to be the optimum scheme of treatment. In contrast to chemotherapeutic preparations, bacteriophages had no effect on normal microflora and did not aggravate dysbiotic disturbances. For this reason, bacteriophages may become one of alternative antimicrobial remedies, selectively affecting infective agents.     

Pathway of galactitol catabolism in Klebsiella pneumoniae.

Cell extracts of galactitol-grown Klebsiella pneumoniae phosphorylate galactitol by means of a phosphoenolpyruvate:galactitol phosphotransferase system. Both the product and authentic L-galactitol-l-P are oxidized with NAD⁺ by a dehydrogenase to yield D-tagatose-6-P, which is phosphorylated with ATP by a kinase to form D-tagatose-1,6-P₂. This ketohexose diphosphate is cleaved by an aldolase to yield dihydroxyacetone-P and D-glyceraldehyde-3-P. Mutants deficient in either the dehydrogenase, kinase, or aldolase failed to grow on galactitol, indicating that the described pathway is of physiological significance in this organism.