Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.     

Drug Resistance of Enteric Bacteria

A nontransferable R₂₁ (TC) factor was obtained by transduction of R₁₀ (TC.CM.SM.SA) with phage epsilon in group E Salmonella. The R₂₁ (TC) factor acquired transmissibility by the normal conjugal process when group E Salmonella strains harboring R₂₁ (TC) factor were infected with wild-type F or R₁₆ (CM) factor. This transmissibility at high frequency was accounted for by the formation of the recombinant F TC and R₁₀ (CM) TC factors. The F TC and R₁₆ (CM) TC factors were genetically the same as the original F and R₁₆ (CM) factors, except for the ability to confer TC resistance. In the transduction of F TC factor with phage P1, a dF TC (d: defective) factor was obtained that was defective in many F properties, such as the ability to introduce host chromosome and produce male substance, but was capable of transducing TC resistance (dF TC-infection) at low frequency.     

Drug Resistance of Enteric Bacteria IX. Distribution of R Factors in Gram-negative Bacteria from Clinical Sources

Many isolates belonging to the Enterobacteriaceae were collected in 1965 from the inpatients at geographically scattered hospitals in Japan. Among 2,650 Shigella strains examined, 58.4% were found to be drug-resistant; 95.0% of these resistant strains were multiply resistant. Among 434 resistant strains examined, 81% carried R factors that were transferable by cell-to-cell contact. Of 160 isolates of other enteric bacteria, drug-resistant strains included 84.2% of the Escherichia coli, 93.0% of the Klebsiella, and 90.0% of the Proteus cultures. Among these resistant strains, 70.3% of the E. coli, 66.7% of the Klebsiella, and 52.0% of the Proteus were multiply resistant. Of these resistant strains, 84.0% of the E. coli, 88.0% of the Klebsiella, and 50.0% of the Proteus strains carried R factors. These results indicate that R factors are widespread among gram-negative bacteria of clinical significance.     

Drug Resistance of Enteric Bacteria VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

Long-Term Shifts in Patterns of Antibiotic Resistance in Enteric Bacteria

Several mechanisms are responsible for the ability of microorganisms to tolerate antibiotics, and the incidence of resistance to these compounds within bacterial species has increased since the commercial use of antibiotics became widespread. To establish the extent of and changes in the diversity of antibiotic resistance patterns in natural populations, we determined the MICs of five antibiotics for collections of enteric bacteria isolated from diverse hosts and geographic locations and during periods before and after commercial application of antibiotics began. All of the pre-antibiotic era strains were susceptible to high levels of these antibiotics, whereas 20% of strains from contemporary populations of Escherichia coli and Salmonella enterica displayed high-level resistance to at least one of the antibiotics. In addition to the increase in the frequency of high-level resistance, background levels, conferred by genes providing nonspecific low-level resistance to multiple antibiotics, were significantly higher among contemporary strains. Changes in the incidence and levels of antibiotic resistance are not confined to particular segments of the bacterial population and reflect responses to the increased exposure of bacteria to antimicrobial compounds over the past several decades.     

Drug Resistance of Enteric Bacteria XIII. Distribution of R Factors in Escherichia coli Strains Isolated from Livestock

Escherichia coli strains isolated from 151 swine and 108 fowl, which were kept at the Animal Health Center, Maebashi, Japan, were surveyed for drug resistance and distribution of R factors. All of the swine and 38% of the fowl excreted E. coli strains resistant to tetracycline, chloramphenicol, streptomycin, and sulfanilamide, or certain combinations thereof. Among 278 resistant cultures isolated from swine, 13% were found to be resistant to one antibiotic, whereas 87% were resistant to more than one antibiotic. Among these resistant strains, 40% carried R factors which were transferable by the usual conjugal process. The resistance patterns of these R factors included 36% which were singly resistant and 64% which were multiply resistant. Among 54 resistant cultures isolated from fowl, 24% were singly resistant and 76% were multiply resistant. Of the resistant strains from fowl, 22% carried R factors. The resistance patterns of R factors included 50% of the singly resistant type and 50% which were multiply resistant. In spite of feeding with dairy products containing only tetracycline, a high incidence of multiple resistance was observed in the E. coli strains and the R factors isolated from these animals.     

Synchronous Growth of Enteric Bacteria

Helmstetter and Cummings devised a technique of synchronization in which cells are implanted on a membrane filter and the membrane is subjected to reverse flow of liquid medium. The cells in the effluent stream have predominantly the characteristics of newborn cells. The advantage of this technique is that the population experiences a minimum of physiological stress; hence, the behavior of the synchronous culture should reflect the normal divisional cycle. The disadvantage is that strains other than Escherichia coli B/r cannot be synchronized. We have found that a modification of the method makes it possible to synchronize several strains of E. coli, including both male and female strains, as well as Salmonella typhimurium LT2. The principal difference in technique is a prolonged period (>400 doublings) of cultivation in glucose minimal medium at 30 C and at low density (<5 x 10(6) cells/ml) prior to implantation. This precaution was taken to insure that the bacterial growth population is in a steady state of balanced growth. From the resulting synchronous growth, the distribution of interdivision times has been computed; these distributions have coefficients of variation in the range 0.18 to 0.22 and are not appreciably skewed.     

猪肝沙门菌的分离鉴定及耐药性分析

根据动物产品中沙门菌的分离和耐药性分析,来评估猪肝中沙门菌的危害程度。对猪肝沙门菌采用分离培养,并进行药敏纸片扩散试验法分析,得出猪肝中沙门菌耐药现象严重的结论。从猪肝中所分离出的5株沙门菌都有不同程度的耐药存在,而且多重耐药现象也普遍存在,特别是对庆大霉素、利福平、多粘菌素B等常用药物耐药(耐药率100%),建议近期对猪沙门菌病的治疗用药为阿米卡星、头孢克肟、头胞噻肟。这些提示当用药物进行治疗时,必须在用药前对病原菌进行药敏试验,只有这样才能有针对性地筛选敏感药物,以提高疗效。     

A Propionate-Inducible Expression System for Enteric Bacteria

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P_prpB) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P_prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P_prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P_prpB system exhibited negligible basal expression by addition of glucose to the medium.     

Studies of Drug Resistance and R Factors in Bacteria from Pond-Cultured Salmonids

Considerable fractions of gram-negative bacilli, Aeromonas liquefaciens, A. salmonicida, Pseudomonas, Hafnia and unidentified Enterobacteriaceae, isolated from the intestinal tracts of cultured Amago (Oncorhynchus rhodurus macrostomus) from 23 Amago-ponds and of Yamame (O. masou ishikawae) from one pond as well as the water of 3 ponds in Shiga, Gifu and Nagano Prefectures and Tokyo Metropolis in Japan were found to be multiple-drug-resistant. Of these drug-resistant strains, A. salmonicida and A. liquefaciens carried R factors at high frequencies and to be prevalent in many Amago ponds throughout Japan. These R factors had markers of resistance to SA, SA.TC, SA.SM.CM. All the R factors belonged to fi^– type.     

重症监护病房医院感染菌分布与耐药性分析

了解哈尔滨医科大学附属第一医院重症监护病房(ICU)病原菌的分布特点及耐药性,为临床治疗提供理论依据。对2003年1月至2010年12月ICU患者送检的标本进行培养,用API鉴定系统或VITEK 2 Com-pact全自动细菌鉴定仪进行菌种鉴定及药敏分析,对检出菌株的构成、标本的分布及药敏结果进行分析。共分离出4 197株病原菌,其中革兰阴性杆菌52.1%、革兰阳性球菌22.3%、真菌25.6%;革兰阴性杆菌中非发酵菌占主要地位(37.5%),以鲍曼不动杆菌为主(13.1%);而肠杆菌科细菌则以肺炎克雷伯菌(9.5%)及大肠埃希菌(5.1%)为主;革兰阳性球菌以金黄色葡萄球菌(14.6%)为主,真菌以白假丝酵母菌(11.8%)为主;在检出所有病原菌的标本中,前3位依次为痰液(79.1%)、血液(8.4%)、无菌体液(8.1%);细菌耐药性结果表明,大肠埃希菌及肺炎克雷伯菌产超广谱β-内酰胺酶(ESBLs)阳性率分别为66.4%、56.3%。几种主要病原菌对各类抗菌药物的耐药程度各不相同,但大部分菌株均呈现多重耐药的现象。产酶菌的耐药率普遍高于非产酶菌,哌拉西林/他唑巴坦对产酶菌及非产酶菌的抑菌效果都很明显;美罗培南、亚胺培南和头孢替坦对肠杆菌科细菌耐药率最低均在40%以下,可作为产酶菌的首选药;鲍曼不动杆菌耐药性严重耐药率均在50%以上。耐甲氧西林葡萄球菌(MRS)检出率为70.1%,其中耐甲氧西林金黄色葡萄球菌(MRSA)75.8%,耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)38.7%。万古霉素、替考拉宁和利奈唑烷可作为革兰阳性球菌严重感染的首选药物。ICU患者以呼吸道感染为主,病原菌以鲍曼不动杆菌和金黄色葡萄球菌为主,且多为多重耐药菌。真菌感染率增加且真菌对药物的敏感性好。应动态监测ICU病原菌的流行和耐药情况,从而控制医院内感染,减少耐药菌株的产生。     

The Evolution of Insertion Sequences within Enteric Bacteria

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.     

Enteric Micro-Particles for Targeted Oral Drug Delivery

This work is focused on production of enteric-coated micro-particles for oral administration, using a water-in-oil-in-water solvent evaporation technique. The active agent theophylline was first encapsulated in cellulose acetate phthalate (CAP), a pH-sensitive well-known polymer, which is insoluble in acid media but dissolves at neutral pH (above pH 6). In this first step, CAP was chosen with the aim optimizing the preparation and characterization methods. The desired release pattern has been obtained (low release at low pH, higher release at neutral pH) but in presence of a low encapsulation efficiency. Then, the CAP was replaced by a novel-synthesized pH-sensitive poly(methyl methacrylate–acrylic acid) copolymer, poly(MMA–AA). In this second step, the role of two process parameters was investigated, i.e., the percentage of emulsion stabilizer (polyvinyl alcohol, PVA) and the stirring power for the double emulsion on the encapsulation efficiency. The encapsulation efficiency was found to increase with PVA percentage and to decrease with the stirring power. By increasing the PVA content and by decreasing the stirring power, a high stable double emulsion was obtained, and this explains the increase in encapsulation efficiency found.     

骨科患者伤口分泌物病原菌分布及耐药性分析

目的:了解我院骨科患者伤口分泌物病原菌分布及其耐药性情况,为临床上对骨科患者合理使用抗生素提供相关理论根据。方法:将2013年2月至2014年8月我院282例术后骨科患者的伤口分泌物标本接种培养,按要求分离纯菌,采用VITEK 2Compact全自动微生物分析仪进行鉴定及药敏试验。结果:282份标本中分离出致病菌186株(65.96%),其中革兰氏阴性球菌94株(50.54%),革兰氏阳性球菌83株(44.62%),真菌9株占4.83%。分离率排在前三位的致病菌分别为阴沟肠杆菌(19.35%),金黄色葡萄球菌(17.20%),表皮葡萄球菌(15.59%)。阴沟肠杆菌、大肠埃希菌、肺炎克雷伯菌对头孢唑林和氨苄西林的耐药率最高,其中阴沟肠杆菌对头孢唑林耐药率高达100%。但未发现主要革兰氏阴性球菌对亚胺培南耐药。革兰氏阳性球菌对青霉素的耐药率较高,但未发现革兰氏阳性球菌对万古霉素耐药。结论:术后骨科患者伤口优势菌种是阴沟肠杆菌,而且耐药性高;临床医生应根据病菌鉴定和药敏分析结果,对不同种类的病原菌使用不同的抗生素进行针对性治疗。     

Incidence of Infectious Drug Resistance Among Lactose-Fermenting Bacteria Isolated from Raw and Treated Sewage

Raw and treated sewage samples were examined for antibiotic-resistant, lactose-fermenting bacteria. Approximately 1% of the total lactose-fermenting bacteria were multiply resistant. Of these organisms, 50% were capable of transferring all or part of their resistance to a drug-sensitive recipient. Only 43% of those isolated on media containing a single antibiotic were capable of resistance transfer, whereas 57% of those recovered on multiple antibiotic plates transferred resistance. R factors conferring resistance to chloramphenicol, streptomycin, and tetracycline; streptomycin and tetracycline; and ampicillin, streptomycin, and tetracycline accounted for 22, 19, and 15%, respectively, of those identified. The data indicate a significant level of infectious drug resistance among the intestinal bacteria of the urban population.     

Rapid Determination of the Presence of Enteric Bacteria in Water

A rapid and sensitive method is described for the detection of bacteria in water and various other natural substrates by the isolation of specific bacteriophage. By the addition of large numbers of the organism in question to the sample, the presence of virulent bacteriophage can be demonstrated in as little as 6 to 8 h. Fecal coliform, total coliform, and total coliphage counts were determined for over 150 water samples from several geographical areas over a period of 2 years. Computer analysis of the data shows a high degree of correlation between fecal coliforms and the coliphage present in the samples. With a high correlation coefficient between fecal coliform and coliphage counts, predictions of the fecal coliforms may be made by enumeration of the phage.