Ampicillin inactivation in the caecum of axenic, gnotoxenic and conventional lambs: interaction with resistant or sensitive Escherichia coli

The fate of orally administered ampicillin was studied in axenic lambs, in gnotoxenic lambs given a complex microflora and a mixture of ampicillin resistant and/or sensitive strains of Escherichia coli, and in conventional lambs. In axenic lambs or animals with a sensitive microflora, antibiotic concentrations of 500-1600 micrograms ml-1 were detected in the intestine, and most of the ampicillin passed through the small intestine and entered the large intestine, within 12-15 h of administration. These antibiotic concentrations were sufficient to decrease the numbers of ampicillin-sensitive E. coli from 10(8)-10(9) bacteria ml-1 to about 10(5)-10(6) bacteria ml-1 by 8 h after ampicillin administration. Second and third doses of antibiotic had no further effect on the bacterial count. Administration of ampicillin to animals hosting ampicillin-resistant E. coli resulted in a significant inactivation of the antibiotic in the intestine. As might be expected there was little reduction in the numbers of these organisms. These results are similar to those observed in conventional lambs hosting resistant E. coli as the dominant colibacillary flora.     

Transepithelial activation of human leukocytes by probiotics and commensal bacteria: Role of Enterobacteriaceae-type endotoxin

The goal of the current study was to clarify whether commercially available probiotics induce greater trans-epithelial activation of human leukocytes than do commensal, food-derived and pathogenic bacteria and to identify the compounds responsible for this activation. Eleven different bacterial strains, and some of their pathogen-associated molecular patterns, were incubated apically on a confluent layer of intestinal epithelial cells (Caco-2), which were basolaterally co-cultured with human mononuclear leukocytes. Only Gram-negative bacteria having Enterobacteriaceae -type endotoxin (commensal Escherichia coli K12, probiotic E. coli Nissle, EPEC) induced basolateral production of TNF-α, IFN-γ, IL 6, 8, and 10. Gram-positive probiotics ( Lactobacillus spp. and Bifidobacterium spp. ) had virtually no effect. In addition, commensals ( Enterococcus faecalis , Bacteroides vulgatus ) and food fermenters ( Lactobacillus spp. ) did not stimulate leukocytes transepithelially. Endotoxin from E. coli and Salmonella enterica Typhimurium induced comparable transepithelial stimulation of leukocytes, but not endotoxin from B. vulgatus or lipoteichoic acid from E. faecalis . Endotoxin-binding agents (polymyxin, colistin) completely abrogated transepithelial activation of leukocytes. Enterobacteriaceae -type endotoxin is a crucial factor in transepithelial stimulation of leukocytes, regardless of whether it is produced by probiotics or other bacteria. Hence, transepithelial stimulation of leukocytes' innate immune response seems to not be linked to the health-promoting effects of probiotics.     

Molecular properties and antibacterial activity of the methyl and ethyl ester derivatives of ampicillin

Ampicillin is a beta-lactam antibiotic that is effective against gram-negative bacteria. Ampicillin has a single carboxyl group (-C(O)OH) within its structure which is suitable for forming ester compounds. Diazomethane and diazoethane were utilized to react with ampicillin to form the methyl and ethyl esters, respectively. The ester derivatives of ampicillin were solubilized together (mole ratio 1:1) in LB media and penicillin resistant Escherichia coli added to measure antibacterial activity. Growth inhibition of bacteria was monitored by optical density after a known time period and with known specific concentrations of the ampicillin esters present. Significant growth inhibition of penicillin resistant bacteria occurred at concentrations of the combined methyl and ethyl ampicillin esters from less than 50 microgram/mL to more than 150 microgram/mL. Molecular properties of the ester compounds were determined. The two ester derivatives showed values of Log BB, Log P, polar surface area, intestinal absorption, and solubility suitable for clinical application. The two ester compounds showed zero violations of the Rule of 5 indicating good bioavailability. The two ester derivatives showed greater intestinal absorbance and greater penetration of the blood brain barrier than the parent ampicillin. Favorable druglikeness was determined for both ester derivatives.     

Study of the inactivation of antibiotics by bacterial colonies

A procedure of bacteria application to disks from the colonies was used for determining antibiotic inactivation in the disks by the bacteria colonies after the disk direct contact with the colonies. Changes in the antibiotic activity in the disks were registered after incubation at 37 degrees C for 2 hours. It was shown that ampicillin resistant strains of E. coli K12 carrying R plasmids and strains of S. typhimurium and S. aureus inactivated the antibiotics in the disks and their population were homogenous in this respect. It is advisable to use the procedure in assaying drug resistance of bacterial populations.     

The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes.

Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.     

The activity of polymyxins against dense populations of Escherichia coli.

The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.     

Characterization of antimicrobial resistance among Escherichia coli isolates from chickens in China between 2001 and 2006

Escherichia coli is a common commensal bacterium and is regarded as a good indicator organism for antimicrobial resistance for a wide range of bacteria in the community and on farms. Antimicrobial resistance of E. coli isolated from chickens from 49 farms in China between 2001 and 2006 was studied. A total of 536 E. coli isolates were collected, and minimal inhibitory concentrations (MICs) of eight antimicrobials were determined by the broth microdilution method. Isolates exhibited high levels of resistance to ampicillin (80.2%), doxycycline (75.0%) and enrofloxacin (67.5%). Relatively lower resistance rates to cephalothin (32.8%), cefazolin (17.0%) and amikacin (6.5%) were observed. Strains were comparatively susceptible to colistin (MIC(50)=1 mug mL(-1)). A marked increase in isolates with elevated MICs for florfenicol was observed over the study period. Therefore, five resistance genes leading to the dissemination of phenicol resistance in the isolates (n=113) with florfenicol MICs>/=32 mug mL(-1) were analyzed. The gene floR was the most prevalent resistance gene and was detected in 92% of the 113 isolates, followed by the cmlA (53%), catA1 (23%) and catA2 (10%) genes. catA3 was not detected in these isolates. Eight isolates with florfenicol MICs=32 mug mL(-1) and one with MIC=64 mug mL(-1) were negative for the floR gene.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

In Vitro Action of Carbenicillin Against Bacteria Isolated from Clinical Material

More than 500 bacteria isolated from patient material were tested against carbenicillin (disodium alpha-carboxybenzylpenicillin) by diffusion and dilution modalities. The same bacteria, which included Pseudomonas aeruginosa, Escherichia coli, Klebsiella-Aerobacter-Enterobacter group, various species of Proteus, Staphylococcus aureus and epiddermidis, enterococci, pneumococci, Streptococcus pyogenes, etc., were examined for susceptibility to other antibiotics commonly used with special emphasis on ampicillin and cephalothin. The responses of pyocine-typed P. aeruginosa were the most remarkable. The majority of these bacteria displayed susceptibility to carbenicillin by both the dilution and the diffusion techniques. The concentrations of this antibiotic used in the laboratory were of the same order of magnitude as that of the other drugs. The laboratory behavior of the other bacteria, toward this new semisynthetic penicillin derivative approximated their response to ampicillin and cephalothin.     

Enhancing the antibiotic antibacterial effect by sub lethal tellurite concentrations: tellurite and cefotaxime act synergistically in Escherichia coli

The emergence of antibiotic-resistant pathogenic bacteria during the last decades has become a public health concern worldwide. Aiming to explore new alternatives to treat antibiotic-resistant bacteria and given that the tellurium oxyanion tellurite is highly toxic for most microorganisms, we evaluated the ability of sub lethal tellurite concentrations to strengthen the effect of several antibiotics. Tellurite, at nM or μM concentrations, increased importantly the toxicity of defined antibacterials. This was observed with both gram negative and gram positive bacteria, irrespective of the antibiotic or tellurite tolerance of the particular microorganism. The tellurite-mediated antibiotic-potentiating effect occurs in laboratory and clinical, uropathogenic Escherichia coli, especially with antibiotics disturbing the cell wall (ampicillin, cefotaxime) or protein synthesis (tetracycline, chloramphenicol, gentamicin). In particular, the effect of tellurite on the activity of the clinically-relevant, third-generation cephalosporin (cefotaxime), was evaluated. Cell viability assays showed that tellurite and cefotaxime act synergistically against E. coli. In conclusion, using tellurite like an adjuvant could be of great help to cope with several multi-resistant pathogens.     

Interactions between Mytilus haemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling

Marine bivalves accumulate large amounts of bacteria from the environment (mainly Vibrionaceae and coliforms). Although persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating haemocytes and haemolymph soluble factors, the mechanisms involved in bacteria-host cell interactions in these invertebrates are largely unknown. In the mussel Mytilus, differences in interactions between haemocytes and different Escherichia coli and Vibrio cholerae strains [E. coli MG155, a wild-type strain carrying type 1 fimbriae, and its unfimbriated derivative, AAEC072 Deltafim; V. cholerae O1 El Tor biotype strain N16961, carrying the mannose-sensitive haemagglutinin (MSHA), and its MSHA mutant] lead to differences in bactericidal activity in the presence of serum. Here we show that different bacteria induced distinct patterns of phosphorylation of mitogen-activated protein kinases (MAPKs), in particular of the stress-activated MAPKs involved in the immune response. Differences in phosphorylation of PKC-like proteins were also observed. The results support the hypothesis that, like in mammalian host cells, different bacteria can modulate the signalling pathways of mussel haemocytes. The lower anti-bacterial activity towards the mutant E. coli strain and wild-type V. cholerae compared with wild E. coli may result from a reduced capacity of activating MAPKs. Moreover, the mutant V. cholerae strain that was the most resistant to the haemocyte bactericidal activity induced downregulation of cell signalling and showed the strongest effect on lysosomal membrane stability, evaluated as a marker of bivalve cell stress. These data suggest that certain bacteria could evade the bactericidal activity of mussel haemocytes through disruption of the host signalling pathways.     

Synergistic effect of ayurvedic pearl preparation on enhancing effectiveness of antibiotics

Studies were carried out with ayurvedic preparations derived from pearl, which include preparations bhasma and pishti. The synergistic effect to reduce the dose of antibiotic was tested against E. coli the test bacterium with ampicillin antibiotic by bore well and disks diffusion methods. It was observed that pearl preparations do not show any antibacterial activity but when used at 200 microg/ml concentration with antibiotics, then even at sub-lethal dose, the antibiotic has effectively shown the results with reduced contact time. The protocol was also tested with the other bacteria like, Pseudomonas aeruginosa. Vibrio cholarae, Salmonella typhi, and Staphylococcus aureus and has shown similar results. The pearl bhasma synergistic effect was also tested with other antibiotics such as erythromycin, kanamycin, and ampicillin.     

肛周脓肿患者病原菌结构和耐药性分析

摘要 目的：分析肛周脓肿患者的病原菌结构和其耐药情况,为临床抗菌药物的选择提供依据。方法：收集医院2019年1月~2020年12月期间收治的324例肛周脓肿患者的脓液标本,在实验室进行菌株的培养、鉴定及药敏试验,记录并分析结果。结果：324例患者均送检标本共分离出病原菌516株,其中革兰阴性菌421株,占81.59%;革兰阳性菌95株,占18.41%。421株革兰阴性菌中,以大肠埃希菌的检出率最高,其次是肺炎克雷伯菌;95株革兰阳性菌中金黄色葡萄球菌检出率最高。在革兰阴性菌中,大肠埃希菌整体耐药率较高,其对氨苄西林、头孢吡肟的耐药率均超过60.00%;肺炎克雷伯菌的耐药率相对较低,仅对头孢曲松、氨苄西林的耐药率超过30.00%。在革兰阳性菌中,金黄色葡萄球菌、表皮葡萄球菌和溶血葡萄球菌对氨苄青霉素、红霉素、环丙沙星和左氧氟沙星的耐药率均较高,超过50%,同时三种葡萄球菌均未检出万古霉素和利奈唑胺的耐药菌株。结论：肛周脓肿患者的病原菌以革兰阴性菌为主,大多为大肠埃希菌和肺炎克雷伯菌,病原菌对不同抗菌药物的耐药性差异较大,应根据药敏结果合理选用抗菌药物。     

Sensitivity to antibacterial preparations of the pseudotuberculosis bacteria isolated on the territory of the Ukraine]

Sensitivity of 92 strains of the causative agent of pseudotuberculosis isolated in the Ukraine was studied with respect to 26 antibacterial drugs. It was found that the strains of the pseudotuberculous bacteria were sensitive to 17 drugs, i.e. benzylpenicillin, ampicillin, carbenicillin, cephaloridin, chloramphenicol, tetracycline, streptomycin, neomycin, monomycin, kanamycin, gentamicin, polymyxin, colistin, furadontin, nalidixic acid, sulfisoxazol and septrin. No differences in the sensitivity of the strains isolated in various districts of the Ukraine and from various sources were found. By their antibiotic sensitivity the strains isolated in the Ukraine did not differ from the cultures isolated in other districts of the USSR and abroad.     

粘菌素对耐碳青霉烯类抗生素鲍曼不动杆菌的抗菌作用

感染耐碳青霉烯的鲍曼不动杆菌(CR-Ab)常与高发病率和死亡率相关联,而可供选择的治疗方案有限,大多基于与粘菌素联用。长期用药导致CR-Ab对粘菌素也产生一定抗性。为了评估含有或不含有粘菌素的不同抗菌组合对从CR-Ab感染患者收集的CR-Ab临床分离株的体外抗菌活性,本研究从本院就诊的患者中收集CR-Ab菌株,通过常量肉汤稀释法(MBD)测定最低抑菌浓度(MICs),通过定性(棋盘法)和定量(即杀菌测试)方法评估各组药物协同活性。结果发现所有菌株均是碳青霉烯类抗性的,且其中两株菌对粘菌素有抗性。棋盘法结果表明含粘菌素的组合在不同处理时间下具有完全协同作用,粘菌素+万古霉素和粘菌素+利福平表现出最高的协同增效作用;不含粘菌素的组合则在35.7%的菌株中观察到完全协同作用。杀菌测试表明粘菌素+美罗培南、粘菌素+替加环素和美罗培南+替加环素组合对粘菌素敏感和低粘菌素抗性的菌株具有杀菌和协同作用,而只有粘菌素+万古霉素和粘菌素+利福平组合表现出持久的杀菌活性。     

大肠杆菌外膜蛋白ompW基因敲除菌的构建及其对两种抗生素的敏感性

探讨大肠杆菌ompW基因敲除后,硫酸新霉素和氨苄青霉素对敲除菌最低抑菌浓度(MIC)和生存率的影响,进而分析OmpW的功能.[方法]运用Red重组技术将大肠杆菌K12染色体上基因ompW敲除,构建缺陷株△ompW.然后分别测定硫酸新霉素和氨苄青霉素对正常菌和△ompW菌的最小抑菌浓度(MIC)及次抑菌浓度(1/2 MIC)下K12和△ompW菌的生存率.[结果]经PCR鉴定和通过提取膜蛋白进行western blot 分析表明,成功获得ompW敲除菌.抗生素分析表明,K12菌对硫酸新霉素的MIC为8.0 μg/mL,生存率为98.0％;△ompW菌对新霉素的MIC为1.7 μg/mL,而其生存率仅为39.0％.而k12对氨苄新霉素的MIC为16.0 μg/mL,△ompW为3.3 μg/mL;1/2 MIC下K12生存率为70.4％,而△ompW为30.3％.[结论]ompW基因缺陷株对两抗生素的敏感性大大增强,表明ompW在细菌抗性方面起着关键作用.     

Incidence and transfer of R-plasmids at a hospital in Saudi Arabia

One hundred and fifty Gram-negative bacteria isolated from patient specimens at King Faisal Specialist Hospital were examined for their ability to transfer antibiotic resistance plasmids to a sensitive Escherichia coli recipient in conjugation and transformation experiments. Agarose gel electrophoresis was used to enumerate and size the R-plasmids found, and Southern DNA hybridization was used to assess similarities between antibiotic resistance plasmids from different bacteria and sources. Of the bacterial isolates tested 65% contained plasmids, 70% of these transferred antibiotic resistance to E. coli, and 40% transferred multiple, linked resistances on R-plasmids. DNA hybridization of these R-plasmids demonstrated widespread similarities between plasmids from different bacterial genera and from different hospital locations. In particular, a gene encoding ampicillin resistance appeared especially widespread, indicating that a transposon may be mediating transmission of this resistance.     

Selective Interaction of Colistin with Lipid Model Membranes

Although colistin’s clinical use is limited due to its nephrotoxicity, colistin is considered to be an antibiotic of last resort because it is used to treat patients infected with multidrug-resistant bacteria. In an effort to provide molecular details about colistin’s ability to kill Gram-negative (G(?)) but not Gram-positive (G(+)) bacteria, we investigated the biophysics of the interaction between colistin and lipid mixtures mimicking the cytoplasmic membrane of G(+), G(?) bacteria as well as eukaryotic cells. Two different models of the G(?) outer membrane (OM) were assayed: lipid A with two deoxy-manno-octulosonyl sugar residues, and Escherichia coli lipopolysaccharide mixed with dilaurylphosphatidylglycerol. We used circular dichroism and x-ray diffuse scattering at low and wide angle in stacked multilayered samples, and neutron reflectivity of single, tethered bilayers mixed with colistin. We found no differences in secondary structure when colistin was bound to G(?) versus G(+) membrane mimics, ruling out a protein conformational change as the cause of this difference. However, bending modulus K_C perturbation was quite irregular for the G(?) inner membrane, where colistin produced a softening of the membranes at an intermediate lipid/peptide molar ratio but stiffening at lower and higher peptide concentrations, whereas in G(+) and eukaryotic mimics there was only a slight softening. Acyl chain order in G(?) was perturbed similarly to K_C. In G(+), there was only a slight softening and disordering effect, whereas in OM mimics, there was a slight stiffening and ordering of both membranes with increasing colistin. X-ray and neutron reflectivity structural results reveal colistin partitions deepest to reach the hydrocarbon interior in G(?) membranes, but remains in the headgroup region in G(+), OM, and eukaryotic mimics. It is possible that domain formation is responsible for the erratic response of G(?) inner membranes to colistin and for its deeper penetration, which could increase membrane permeability.