整合子介导产生超广谱β-内酰胺酶志贺菌的多重耐药研究

目的探讨产生超广谱β-内酰胺酶（ESBL）志贺菌的耐药特性及其耐药机制。方法采用K-B法药敏试验筛选可疑产ESBL志贺菌株;通过改良三维试验、E-test试验及相对水解率测定对可疑产ESBL志贺菌进行鉴定;采用TEM、SHV、CTX-M-1组、CTX-M-2组和CTX-M-9组β-内酰胺酶通用引物进行PCR检测,并对TEM和CTX-M-9组全编码基因引物等PCR扩增产物进行DNA序列分析;对产ESBL志贺菌进行结合传递试验,供体菌和接合子用稀释法进行MIC测定。对ESBL菌株进行Ⅰ类、Ⅱ类和Ⅲ类整合子的多重PCR检测,Ⅰ类整合子可变区PCR扩增产物进行DNA测序,确定耐药基因盒的种类和数量。结果275株志贺菌中有12株为产ESBL志贺菌,其基因型为CTX-M-14和CTX-M-1组;产ESBL志贺菌结合传递试验全部阳性,其结合子只对β-内酰胺类抗生素耐药。12株ESBL志贺菌只含Ⅰ类整合子,整合子可变区含有dfrA17-aadA5耐药基因盒。结论济南地区志贺菌对β-内酰胺类抗生素的交叉耐药由质粒介导,对磺胺类和氨基糖苷类多重耐药由整合子介导。     

肉鸡源多重耐药空肠、结肠弯曲菌的耐药分子特征

目的了解肉鸡源空肠和结肠弯曲菌的耐药谱特征,检测多重耐药菌株Ⅰ类整合子/基因盒、gyrA基因突变位点、tetO基因、23S rRNA突变位点的分子特征。方法利用PCR检测弯曲菌Ⅰ类整合子/基因盒的存在情况;利用MAMA PCR技术检测弯曲菌gyrA基因第257位碱基的突变情况;针对弯曲菌23S rRNA的V区2075突变位点检测突变菌株。结果多重耐药菌株占分离株的94.5%。146株多重耐药空肠和结肠弯曲菌中Ⅰ类整合子检出率为98.6%,有78株菌株检出3种基因盒,1 000 bp+750 bp+500 bp+250 bp为主要谱型,所占比例为92.3%;有131株在gyrA喹诺酮类耐药决定区发生突变,突变率为92.9%。127株四环素耐药弯曲菌tetO基因的检出率为95.3%。81株红霉素耐药菌株中,23S rRNA的V区2075处突变发生率为96.3%。结论空肠和结肠弯曲菌分离株携带aadA2耐药基因盒,与氨基糖苷类药物的耐药性相关;gyrA基因突变、tetO基因的携带以及23S rRNA突变,与弯曲菌对喹诺酮、四环素和大环内酯类耐药密切相关。     

医院污水中大肠埃希菌的耐药谱及Ⅰ类整合子分析

目的调查医院污水中多重耐药菌污染情况及其耐药基因和Ⅰ类整合子携带情况。方法采集河南省郑州大学第一附属医院污水,使用16S rRNA测序和微量生化反应管鉴定细菌种属;采用PCR扩增耐药基因和Ⅰ类整合子基因盒,计算其检出率。结果根据16S测序和生化反应共分离鉴定出23株大肠埃希菌。药敏试验显示23株大肠埃希菌对四环素的耐药率为62.5%(15/23),复方新诺明为56.5%(13/23),氨苄西林为56.5%(13/23)。所有菌株均对美罗培南和亚胺培南敏感。大肠埃希菌多重耐药率为56.52%(13/23)。PCR检测耐药基因携带率aadA1基因为100%,tetA基因为95.8%,aac(6')-Ib-cr基因为79.2%,CTX基因为70.8%,tetB基因为63.5%,Qnrs基因为58.3%,SHV基因为54.2%,qepA基因为41.7%,Sul基因为35%。所有菌株均不携带KPC基因、IMP基因和NDM1基因。有21株细菌检测到Ⅰ类整合子,其中11株细菌的整合子携带空基因盒。整合子携带的大多数基因盒编码DfrA和aadA基因,有1株细菌的基因盒编码OXA-1基因。结论医院污水中的大肠埃希菌存在多药耐药现象且Ⅰ类整合子携带率高,应引起高度重视。     

大肠埃希菌中新的耐药基因盒aadA23的变异

目的基于整合子-细菌耐药系统在细菌耐药机制中的重要作用,对成人腹泻患者的大肠埃希菌Ⅰ类整合子阳性菌株携带的耐药基因盒的基因特征进行分析.方法应用聚合酶链反应（PCR）检测Ⅰ类整合酶基因intⅠ阳性菌株并对其整合的耐药基因进行测序及用生物信息软件对序列进行分析.结果5株Ⅰ类整合酶基因阳性菌株的耐药基因盒PCR扩增得到1009 bp的产物.序列分析结果表明,1009 bp序列含有780 bp的开放阅读框,与已知的aadA23和aadA21分别有99.6%和99.5%的相似性,与aadA5只有66.4%相似,为对氨基糖苷类抗菌药物壮观霉素、链霉素产生耐药的基因盒,建议命名为aadA23b.结论随着选择环境不同,整合子整合的基因盒会发生变异,提示我们要用分子生物学的手段从基因水平分析耐药基因的遗传与变异.     

天津地区福氏志贺菌整合子携带及耐药性研究

目的了解天津地区不同年份福氏志贺菌血清型分布,整合子携带情况,耐药性及其变化趋势。方法采用血清学方法对天津地区不同年份分离的56株福氏志贺菌进行分型;PCR扩增整合子整合酶及可变区,并测序;采用K-B法测定其对16种抗菌药物的敏感性。结果 1981~1983年分离株福氏志贺菌1类整合酶阳性率为87.50%(28/32),其中27株可变区含氨基糖苷类药物耐药基因aadA;2009~2011年分离株福氏志贺菌1类整合酶阳性率为91.67%(22/24),可变区及3’末端扩增均阴性。1981~1983年分离株福氏志贺菌2类整合酶均阴性;2009~2011年分离株福氏志贺菌2类整合酶阳性率79.17%(19/24),可变区含dfrA1、sat1、aadA1,介导对甲氧苄啶、链丝菌素和链霉素耐药。有17株福氏志贺菌1、2类整合酶均阳性。1981~1983年分离株福氏志贺菌对四环素、链霉素、氯霉素及甲氧苄啶/磺胺甲噁唑耐药率高,多重耐药率为65.63%;2009~2011年分离株福氏志贺菌对氨苄西林、链霉素、甲氧苄啶/磺胺甲噁唑、哌拉西林和四环素耐药率高,多重耐药率为83.33%。结论 1、2类整合子广泛存在于天津地区福氏志贺菌中,其中1类整合子3’保守末端可能缺失或存在变异。2009~2011年分离株福氏志贺菌对抗菌药物耐药性较1981~1983年分离株增强,多重耐药率增高。福氏志贺菌优势血清型发生转变,血清型分布呈现多样化。     

整合子及相关基因盒在临床分离铜绿假单胞菌中的分布

目的检测Ⅰ类、Ⅱ类和Ⅲ类整合子及Ⅰ类整合子相关基因盒在铜绿假单胞菌临床分离株中的分布,分析整合子对细菌耐药性的影响。方法用纸片法对62株临床分离铜绿假单胞菌进行药敏试验;应用多重PCR法检测62株铜绿假单胞菌Ⅰ类、Ⅱ类和Ⅲ类整合子;对Ⅰ类整合子阳性菌进行整合子相关基因盒检测。结果62株菌中有40株(64.5%)含有Ⅰ类整合子,1株(1.6%)含有Ⅱ类整合子;没有检测到Ⅲ类整合子阳性菌。在Ⅰ类整合子阳性菌中,有26株携带Ⅰ类整合子相关基因盒(65.0%);分离自同一科室的部分菌株携带大小相同的基因盒;Ⅰ类整合子阳性菌株的耐药率高于整合子阴性的菌株。结论Ⅰ类整合子及整合子相关基因盒在铜绿假单胞菌临床菌株中分布广泛,整合子在细菌耐药中发挥作用。     

临床痢疾志贺菌整合子检测及耐药基因盒序列分析

目的 检测印度东部1988、1995和2002年部分临床分离痢疾志贺菌中细菌耐药关系密切的1、2、3类整合酶基因及整合子携带的耐药基因盒的分布,分析整合子系统对志贺菌耐药的影响.方法 纸片扩散法检测实验菌株对药物的敏感性;应用PCR方法对16株临床耐药菌株进行1、2、3类整合酶基因(intI)筛选,对阳性样本可变区基因盒序列进行鉴定分析.结果 所有16株菌均耐4种及4种以上药物,包括β-内酰胺类、氨基糖苷类、四环素类、磺胺类、氯霉素类和喹诺酮类.13株菌检出1类整合酶基因,全部菌株含2类整合酶基因,即发现同时存在两种整合子结构菌株,未检测到3类整合酶基因.1类整合酶插入基因盒以blaara30-aadAl基因家族为主,分别对β-内酰胺类抗生素、链霉素、壮观霉素耐药;2类整合酶插人基因盒以dfrAl-satl组合为主,分别对甲氧苄氨嘧啶、链丝菌素耐药,同时在4株菌中发现dfrAl-satl-aadAl基因盒组合.结论 2类整合子普遍存在于临床志贺菌中.整合子与志贺菌的多重耐药具有密切相关性.     

基因盒-整合子系统与志贺菌耐药

随着抗生素的广泛应用,志贺菌的耐药特别是多重耐药问题日趋严重.具有捕获及表达外来基因能力的整合子(integron)系统作为新的细菌耐药机制,正越来越受关注.根据整合酶int Ⅰ的不同,整合子可分为6类.其中1,2,3类整合子研究较多,并已被证明与细菌的耐药性有关.最近研究表明,2类整合子是志贺菌中最常见的整合子.本文就基因盒-整合子系统的概念、结构及与志贺菌耐药相关性等进行综述.     

屠宰生猪多重耐药沙门菌I类整合子与耐药基因的检测

目的了解屠宰生猪沙门菌分离株多重耐药与I类整合子及耐药基因的携带关系。方法采用K-B纸片法对屠宰生猪78株沙门菌分离菌株进行22种抗生素敏感试验;应用PCR技术对沙门菌分离菌株进行I类整合子及耐药基因检测。结果78株沙门菌分离株中有41株(52.56%)对2种以上抗生素耐药,属于多重耐药株,强力霉素-四环素-卡那霉素-氯霉素是主要多重耐药谱;41株多重耐药菌中有19株(46.34%)携带I类整合子,tetB、aph(3)-IIa和Flor基因分别检出最高。结论沙门菌多重耐药性与整合子的携带之间关系密切,耐药表型测定结果与耐药基因检测结果一致。     

铜绿假单胞菌整合子检测的相关性研究

目的调查天津地区铜绿假单胞菌（Pseudom onas aeruginosa,PA）的整合子流行情况,并分析整合子与PA耐药的相关性。方法收集53株天津地区临床分离的耐碳青霉烯PA,K-B法与MH肉汤微量稀释法测定其药敏情况,并用PCR法扩增整合子的整合酶基因,对阳性PCR产物采用HinfⅠ内切酶作限制片段长度多态性（RFLP）分析进行整合子分类。结果 53株耐碳青霉烯PA对哌拉西林等14种抗菌药的耐药率从30%～100%。18.9%（10/53）的PA中检出整合子;PCR-RFLP结果显示均为Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子。结论Ⅰ类整合子较广泛地存在于天津区耐碳青霉烯铜绿假单胞菌中,整合子与PA的耐药和多重耐药具有相关性。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.