Transfer of kanamycin resistance among familial strains of Enterobacteriaceae isolated from a chronic bacterial carrier of Salmonella schottmuelleri

Escherichia coli, Enterobacter aerogenes and S. schottmuelleri were isolated from the large intestine of a bacteriocarrier. E. coli and E. aerogenes strains proved to be resistant to a number of antibiotics. Plasmids were detected in DNA preparations obtained from E. coli strains. After the hybridization of these E. coli strains with E. coli C600 5K and S. schottmuelleri at 28 degrees C the transfer of resistance to kanamycin was found to occur. From some of the transconjugates thus obtained resistance to kanamycin was transferred to E. aerogenes. This resistance was found to be controlled by the plasmid with a molecular weight exceeding 2 Md. The fact that S. schottmuelleri in the carrier's body retained their sensitivity to antibiotics can be explained by the absence of the transfer of plasmid Kmr at a temperature exceeding 28 degrees C and by the existence of the infective agent in an ecological niche other than that of E. coli.     

Resistance to imipenem, cefepime, and cefpirome associated with mutation in Omp36 osmoporin of Enterobacter aerogenes

Enterobacter aerogenes develops increased multidrug resistance via a functional alteration of outer-membrane permeability associated with a decrease in porin function. We have sequenced the gene coding the major porin of Enterobacter aerogenes, omp36. The sequence shows a high similarity with the Klebsiella pneumoniae ompK36 gene and is closely related to the enterobacterial OmpC family. Sequence analysis of several Omp36 issued from clinical strains indicated variability in putative cell-surface exposed domains. Interestingly, substitution Gly112Asp was observed in the conserved eyelet L3 region of the porin produced by two strains, C and 3. This substitution is associated with a high general beta-lactam resistance observed in these isolates and with alteration of pore properties previously described in strain 3 porin [Mol. Microbiol. 41 (2001) 189]. This is the first genetic identification of impermeability-mediated resistance to beta-lactams in various clinical E. aerogenes strains.     

近三年产气肠杆菌临床感染情况分析

目的监测嵊州市人民医院近三年产气肠杆菌对常用抗生素耐药情况。方法对嵊州市人民医院2013年至2015年间收集的产气肠杆菌临床科室分布情况进行统计,并做临床常用抗生素耐药性分析,用WHONET 5.4软件进行统计学分析。结果分离得到的产气肠杆菌主要来源于神经外科、重症医学科和呼吸内科的痰液和尿液标本。药敏结果显示其对第一、二代头孢类抗生素耐药率普遍较高:对头孢唑啉几乎全耐药,对头孢呋辛耐药率约为55%;第四代的头孢吡肟能明显抑制产气肠杆菌生长,2013年到2015年对头孢吡肟耐药率分别为8.18%、9.14%和10.74%;对氨基糖苷类抗生素庆大霉素和丁胺卡那,以及碳青霉烯类抗生素亚胺培南和美罗培南具有很高的敏感性。结论嵊州市人民医院院感科近年间通过对产气肠杆菌临床用药的合理监测,及时向临床医生反馈微生物实验室的耐药结果,避免了抗生素滥用,使得其对抗生素耐药率未出现明显提高。氨基糖苷类抗生素和碳青霉烯类抗生素对其仍具有很高的疗效,应继续通过药敏试验加强对其耐药性监测,指导临床合理用药。     

The purification and properties of a penicillinase whose synthesis is mediated by an R-factor in Escherichia coli

1. The penicillinase (beta-lactamase) from Escherichia coli strain TEM has been purified and its activity against a range of penicillin and cephalosporin derivatives measured. 2. The enzyme shows little resemblance to penicillinases from Bacillus cereus, Bacillus licheniformis and Staphylococcus aureus. 3. The molecular weight of the enzyme is 16700+/-5%, which is about half the value obtained for other penicillinases. 4. The enzyme is most similar in properties to a crude preparation of a penicillinase from Klebsiella (Aerobacter) aerogenes, but clearly different from crude enzyme preparations from other strains of E. coli. 5. Since penicillinase synthesis in E. coli strain TEM is mediated by an R-factor known to infect many other species of Enterobacteriaceae, the appearance of similar enzymes in other Gramnegative species is not surprising.     

Siderophore-mediated strategies of iron acquisition by extraintestinal isolates of Enterobacter spp

A total of 89 examined Enterobacter isolates belonging to three species: E. cloaceae, E. aerogenes and E. sakazakii, produced iron chelators detected in universal CAS assay. In chemical assays the strains were shown to excrete mostly catecholate (88 strains) and hydroxamate (42 strains) type of siderophores. Forty-one strains produced both catecholate and hydroxamate siderophores whereas one isolate produced only hydroxamate. Besides, the isolates were screened for genes coding for another siderophore: yersiniabactin. The genes for biosynthesis and uptake of yersiniabactin are located on the high-pathogenicity island (HPI) of Yersinia spp. The presence of three marker genes irp1, irp2 and fyuA was estimated by polymerase chain reaction. Two strains: E. aerogenes and E. cloaceae possessed irp1, irp2 and fyuA genes. PCR products of irp1, irp2 and fyuA were of 240, 280 and 780 bp, respectively.     

Cadmium resistance in some members of Enterobacteriaceae

Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16), and Serratia marcescens (16) were screened for Cd resistance or sensitivity. Only one strain each of these was resistant to high levels (25 n moles/0.05 ml) CdCl2. The Minimal inhibitory concentration (MIC) of sensitive strains ranged from 0.8-5 micrograms/ml. All the resistant strains were simultaneously resistant to a number of antibiotics. Treatment with sodium dodecyl sulfate eliminated resistance to Cd and to some antibiotics.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

Transposon Tn5 encodes streptomycin resistance in nonenteric bacteria.

Strains of Caulobacter crescentus, Pseudomonas putida, Acinetobacter calcoaceticus, Rhizobium meliloti, and Rhodopseudomonas sphaeroides carrying the kanamycin resistance-encoding transposon Tn5 were 15 to 500 times more resistant to streptomycin than transposon-free strains. The streptomycin resistance determinant, which is separable from the kanamycin resistance determinant of Tn5, was not expressed in Escherichia coli or Klebsiella aerogenes.     

Periodontal disease as reservoir for multi-resistant and hydrolytic enterobacterial species

AIMS: This investigation aimed to isolate enteric rods from subgingival sites of patients presenting chronic periodontitis lesions, and to assess antimicrobial resistance and expression of hydrolytic enzymes. METHODS AND RESULTS: Enterobacteriaceae were isolated from 20% patients, and assayed for antimicrobial susceptibility and hydrolytic enzymes with specificity to different substrates. Isolates comprised seven Enterobacter cloacae (43.75%), five Serratia marcescens (31.25%), one Klebsiella pneumoniae (6.25%), one Enterobacter aerogenes (6.25%), one Pantoea agglomerans (6.25%), and one Citrobacter freundii (6.25%). Gelatinase activity was observed for 75% strains; caseinase and elastase was produced by six and two strains, respectively. DNase, lecithinase and lipase were expressed by S. marcescens. Most of strains were resistant to ampicillin (93.75%) and amoxicillin/clavulanic acid (81.25%). The majority of strains were susceptible to cephalosporins and aztreonam. Enterobacteria remained susceptible to imipenem, streptomycin and fluoroquinolones. Resistance to gentamicin, amikacin, sulfamethoxazole/thrimethoprim, tetracycline, and chloramphenicol were also observed. Eight strains presented multiple drug resistance. CONCLUSIONS: Subgingival sites from periodontal diseases contain multi-resistant and hydrolytic enzyme-producing enterobacteria that may contribute to overall tissue destruction and spreading. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterobacteria isolated from patients generally considered as healthy individuals poses periodontal diseases as reservoir for systemic infections particularly in immunocompromised and hospitalized hosts.     

2011～2015年医院呼吸内科常见革兰阴性菌耐药性分析与临床用药探讨

目的：分析2011～2015年我院呼吸内科常见革兰阴性菌的分布情况及耐药性特点,为规范抗生素的合理使用提供依据。方法选用法国梅里埃公司生产的ID 32GD试剂盒,应用ATB Expression全自动分析系统对细菌进行鉴定与药敏试验。结果2011～2015年在我院呼吸内科常见的病原菌分别为肺炎克雷伯菌（31．0％）、大肠埃希菌（21．2％）、铜绿假单胞菌（23．2％）与鲍曼不动杆菌（14．8％）。肺炎克雷伯菌对碳青霉烯类的耐药率几乎为0,对单纯的头孢菌素耐药率相对较高,但对含β‐内酰胺酶抑制剂的头孢菌素类以及阿米卡星有较强的抗菌活性;大肠埃希菌对哌拉西林耐药率高,但对含酶青霉素类有较强的抗菌活性,对碳青霉烯类的耐药率连续5年来均为零;铜绿假单胞菌对磺胺类耐药性较高,对头孢三代、头孢四代及含酶青霉素类与含酶头孢类均有较强的抗菌活性,但敏感性呈逐年下降趋势;鲍曼不动杆菌的耐药率呈逐年上升趋势,仅对含酶的头孢哌酮／舒巴坦敏感（约80％）。结论医院呼吸内科革兰阴性菌仍以肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌为主。鲍曼不动杆菌耐药性逐年增长的问题已日趋严重,相关部门应定期监测细菌耐药趋势,指导临床合理用药,特别是合理应用抗生素以避免细菌耐药及减少细菌耐药性的发生。     

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.     

Inhibitors of antibiotic efflux pump in resistant Enterobacter aerogenes strains

Enterobacter aerogenes, a nosocomial pathogen, is frequently exhibiting multidrug resistance mechanisms associated with a change in membrane permeability. In clinical isolates, active efflux plays a prominent role in antibiotic resistance. We report here the effect of three unrelated compounds that are able to restore a noticeable antibiotic susceptibility to resistant strains. The targeting of various parameters which contribute to the efficacy of the efflux mechanism, such as energy, flux selectivity, or functional assembly of the membrane complex, increases the intracellular chloramphenicol concentration in resistant isolates.     

Comparison of the Substrate Specificities of the β-Lactamases from Klebsiella aerogenes 1082E and Enterobacter cloacae P99

A potent beta-lactamase (EC 3.5.2.6) produced by a strain of Klebsiella aerogenes (K. pneumoniae), 1082E, isolated from a hospital patient, has been examined. Its properties were different from those of most gram-negative beta-lactamases previously reported. The enzyme has been partly purified, and its activity against a range of substrates has been compared with that of the enzyme from Enterobacter cloacae (Aerobacter cloacae) P99. The K. aerogenes enzyme, although predominantly a penicillinase, had a wide range of specificity. In addition to hydrolyzing the cephalosporins, it attacked the normally beta-lactamaseresistant compounds methicillin and cloxacillin as well as cephalosporin analogues with the same acyl substituents. The results obtained with the E. cloacae enzyme confirmed its cephalosporinase activity and showed that, unlike the enzyme from K. aerogenes, it was relatively inactive against the penicillins.     

外科手术切口感染大肠埃希菌的耐药性分析

目的分析外科手术切口中大肠埃希菌的感染率及耐药性,为临床合理用药提供科学依据。方法对2009年6月至2010年9月外科患者手术切口标本中分离的大肠埃希菌耐药情况进行分析,并对产超广谱β-内酰胺酶（ESBLs）菌株进行表型确证试验。结果 140株大肠埃希菌中,产ESBLs菌株的检出率为42.14%;产ESBLs菌株对一～四代头孢菌素、广谱青霉素、氟哇诺酮类及氨基糖苷类抗菌药物具有较高的耐药率;非产ESBLs菌株对青霉素类以外的抗菌药物具有较高的敏感率;产ESBLs菌株对大多数抗菌药物的耐药率明显高于非产ESBLs菌株（P〈0.05）。结论外科手术切口术后大肠埃希菌的感染较严重,且产ESBLs菌株多药耐药明显,临床应加强监测与控制。     

Isolation and Characterization of a Pseudomonas Strain Producing Glutaryl-7-Aminocephalosporanic Acid Acylase

Several screening methods were developed for the selection of Pseudomonas strains capable of hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. An isolate exhibiting high acylase activity, designated BL072, was identified as a strain of Pseudomonas diminuta. It grew optimally at pH 7 to 8 and at a temperature of 32 to 40°C, but acylase activity was highest when the strain was grown at 28°C. Mutants of BL072 were generated by nitrosoguanidine treatment and screened for increased production of glutaryl-7-aminocephalosporanic acid acylase. A superior mutant gave a fourfold increase in acylase titer. The cell-associated acylase had similar activities against various glutaryl-cephems but had undetectable activity against cephalosporin C. This acylase may prove useful for the conversion of cephalosporin C to 7-aminocephalosporanic acid.     

Imipenem resistance in Enterobacter aerogenes is associated with derepression of chromosomal cephalosporinases and impaired permeability

Enterobacter aerogenes mutants with high-level resistance to imipenem were studied. They were derived from strains characterized by stable over-production of a class-I beta-lactamase. This enzyme (pI = 8.2) exhibited high affinity toward imipenem and hydrolysed the drug slowly. Imipenem-resistant mutants lacked a major 43-kDa outer membrane protein and displayed decreased permeability to cephaloridine. Introduction of a plasmid coding for the regulatory ampD gene abolished beta-lactamase production and rendered the mutants susceptible to imipenem.     

Protocatechuate is not metabolized via catechol in Enterobacter aerogenes.

Protocatechuate is generally metabolized in bacteria by direct oxygenative cleavage to produce beta-carboxymuconate. An exception to this pattern has been suggested by reports that protocatechuate might be metabolized by nonoxidative decarboxylation to catechol in Enterobacter aerogenes. In the present investigation, analysis of mutant strains indicated that this proposed pathway did not make a significant contribution to protocatechuate metabolism in E. aerogenes because mutations blocking catechol metabolism did not impair protocatechuate utilization. In addition, all the enzymes required for the oxygenative cleavage of protocatechuate and its further metabolism were induced in E. aerogenes during protocatechuate metabolism, and mutations inactivating this oxygenative pathway prevented protocatechuate degradation. The strains of E. aerogenes examined exhibited broad specificities of inductive control over genes associated with protocatechuate and catechol metabolism; it appears that a number of metabolites may trigger the expression of these genes.     

牛蒡根际可培养细菌多样性和镉耐性初步分析

从牛蒡根际土壤中分离可培养细菌,进行多样性分析,并对镉耐受性菌株进行筛选及其抗性和种群多样性进行了分析。限制性内切酶多态性分析显示,分离的菌株可分为9个操作分类单元(OUT),分别属于变形菌门、厚壁菌门和放线菌门,分属于6个科,9个属,其中隶属于肠杆菌属、芽胞杆菌属和假单胞菌属的是优势物种。分离到的耐镉菌株分别属于Bacillus subtilis、Enterobacter aerogenes、Enterobacter ludwigi、Klebsiellasp.、Pectobacterium carotovorum、Pseudomonassp.,而Pectobacterium carotovorumNP22、Enterobacter ludwigii NP23、Pseudomonassp.NP39三菌株可在Cd2+浓度为400 mg/L固体培养基上生长。     

潜在多重耐药患者肠道内乳杆菌的分离与鉴定

目的从临床潜在多重耐药患者粪便中分离获得乳杆菌,完成种属鉴定并分析其临床意义。方法采集临床患者粪便,在MRS和含碳酸钙的LBS固体培养基上分离培养,挑取目的菌落反复分离获得纯化菌株,利用生化鉴定方法进行种属鉴定。结果 45份样本共得到7株乳杆菌,其中植物乳杆菌5株、鼠李糖乳杆菌2株。结论研究发现,分离到的植物乳杆菌和鼠李糖乳杆菌可能具有更强的耐受性与生物学活性。分离获得的植物乳杆菌和鼠李糖乳杆菌有待日后对其开展特性、耐受性及安全性等研究,以便对分离的菌株有更深入的认识。