临床分离4544株革兰阴性杆菌的耐药分布与变迁

目的了解玉溪市革兰阴性杆菌的耐药分布与变迁,为临床用药提供依据。方法对玉溪市人民医院1999—2004年临床各科送检的各类标本中培养分离出的4544株革兰阴性杆菌作回顾性分析。结果9种（属）细菌对25种药物的药敏结果耐药率〉50％者72种次（40．9％）,〈20％者44种次（25．0％）。总的耐药情况为不动杆菌和阴沟肠杆菌最高,铜绿假单胞菌和大肠埃希菌次之,甲型副伤寒沙门菌和福氏志贺菌最低。亚胺培南对多种细菌有较高的敏感覆盖率。有4种细菌对10种抗菌药物耐药率的上升具有临床意义（P〈0.01或P〈0．05）,其中以甲型副伤寒沙门菌对氟喹诺酮类上升最为显著。结论临床分离革兰阴性杆菌的耐药形势十分严峻,应定期监测区域内细菌的耐药变化．指导临床合坪用药。     

Antigen sharing of Salmonella typhi non-flagellar proteins with other salmonellae and some shigellae and Escherichia coli

Cathodal moving protein components were identified in agarose gel electrophoresis of the Veronal buffer extract of a non-motile strain of S. typhi (8393, Colindale). Rabbit antiserum was raised against these cationic proteins; it had both agglutinating and precipitating activity. A total of 80 salmonella strains belonging to serogroups A, B, C1, C2, D, E1 and E2 including 26 S. typhi and 10 S. paratyphi A were tested against this antiserum in a slide agglutination test; all strains were agglutinated. Among 94 other bacterial strains tested, the antiserum agglutinated all 16 strains of Shigella flexneri, 2 of 5 Shigella sonnei, 5 of 34 E. coli and 1 of 8 Citrobacter species. These results show that there is antigenic sharing of the non-flagellar proteins of S. typhi with many other salmonellae as well as with some shigellae and E. coli.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.     

6种沙门菌单克隆抗体的制备和效能评价

目的：制备稳定、特异、高亲和性的分别针对甲型副伤寒沙门菌、乙型副伤寒沙门菌、丙型副伤寒沙门菌、肠炎沙门菌、伤寒沙门菌和猪霍乱沙门菌的单克隆抗体。方法：用甲醛灭活的菌液抗原免疫BALB／c小鼠,取脾细胞与SP2／0骨髓瘤细胞融合;用灭活的菌液包被酶标板,ELISA筛选阳性克隆株,建立细胞系;选取高效分泌杂交瘤细胞,常规制备腹水并纯化,进行单抗特异性与亲和性评价。结果：筛选得到分泌6种沙门菌相应单克隆抗体的杂交瘤细胞株,获得高亲和性单抗;所有单抗与大部分病原菌（包括7种沙门菌、3株志贺菌、2株李斯特菌、4株致病性大肠杆菌、2株霍乱弧菌）无交叉反应,但由于同类型O抗原的广泛分布,抗乙型副伤寒沙门菌单抗与鼠伤寒沙门菌、抗伤寒沙门菌单抗与肠炎沙门菌有明显的交叉反应。结论：沙门菌单抗的制备,为感染性腹泻的监测、诊断奠定了基础。     

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.     

Frequency of Escherichia coli O157:H7 isolation from stool specimens

During a 6-month period, 7252 faeces specimens were examined for Escherichia coli serotype O157:H7. Forty-nine specimens (0.7%) from 19 patients yielded this organism. Escherichia coli O157:H7 was the third most frequently isolated bacterial pathogen, following Campylobacter jejuni and (or) Salmonella sp. Although regional variation between laboratories determined whether Campylobacter jejuni or Salmonella was the primary bacterial pathogen isolated, E. coli O157:H7 was consistently isolated more frequently than either Shigella or Yersinia enterocolitica.     

Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria

A 32P-labeled fragment of DNA, encoding the major part of the chromosomal ampC beta-lactamase gene of Escherichia coli K-12, was used as a hybridization probe for homologous DNA sequences in colonies of Neisseria gonorrhoeae, Pseudomonas aeruginosa, and different enterobacterial species. The ampC probe detected the presence of homologous DNA sequences in clinical isolates of E. coli, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Salmonella typhimurium, Serratia marcescens, and P. aeruginosa. No hybridization was found with N. gonorrhoeae colonies. In Southern blotting experiments the ampC probe hybridized to chromosomal DNA fragments of the same size in all enterobacterial species tested. However, the degree of hybridization differed with DNA from different species. DNA from the Shigella species strongly hybridized to the ampC probe. Furthermore, antibodies raised against purified E. coli K-12 ampC beta-lactamase precipitated beta-lactamases from the Shigella species, suggesting extensive sequence similarities between the ampC genes of these genera. The production of chromosomal beta-lactamase in S. sonnei increased with increasing growth rate similar to E. coli K-12. This growth rate response was abolished in two beta-lactamase-hyperproducing S. sonnei mutants, which thus seem similar to E. coli K-12 attenuator mutants. We propose that both the structure and regulation of the chromosomal beta-lactamase genes are very similar in E. coli and in S. sonnei.     

Comparison of the tryptophan synthetase alpha-subunits of several species of Enterobacteriaceae

Creighton, T. E. (Stanford University, Stanford), D. R. Helinski, R. L. Somerville, and C. Yanofsky. Comparison of the tryptophan synthetase alpha subunits of several species of Enterobacteriaceae. J. Bacteriol. 91:1819-1826. 1966.-The tryptophan synthetase alpha subunits of Escherichia coli K-12, E. coli B, Shigella dysenteriae, Salmonella typhimurium, and Aerobacter aerogenes have been purified and their structures compared. Each of these alpha subunits exhibits a sedimentation coefficient of about 2.7S. Peptide patterns of trypsin plus chymotrypsin digests of the alpha subunits have indicated that all of the alpha subunits have peptide regions in common. The patterns of E. coli K-12, E. coli B, and S. dysenteriae alpha subunits appear to be nearly identical, whereas the alpha subunits from S. typhimurium and A. aerogenes differ from those of E. coli and from each other. It has also been shown that the E. coli structural gene for the alpha subunit is translated identically in E. coli and S. typhimurium.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Bafoudiosbulbins A, and B, two anti-salmonellal clerodane diterpenoids from Dioscorea bulbifera L. var sativa

Two clerodane diterpenoids, Bafoudiosbulbins A 1, and B 2, together with five known compounds: tetracosanoic acid, 1-(tetracosanoyl)-glycerol, trans-tetracosanylferulate, beta-sitosterol and 3-O-beta-D-glucopyranosyl-beta-sitosterol were isolated from the tubers of Dioscorea bulbifera L. var sativa. Their structures were established by spectroscopic methods (1D and 2D-NMR, MS) and X-ray crystallographic diffraction analysis of compound 1. The CH2Cl2-soluble portion of the crude extract and the two clerodanes were screened for anti-bacterial activity using both agar diffusion and broth dilution techniques against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B. They both showed significant activities against P. aeruginosa, S. typhi, S. paratyphi A and S. paratyphi B.     

Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases

Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous ( approximately 15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5alpha (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

Radiation Survival of Food Pathogens in Complex Media

When 15 bacterial species representing genera associated with food-borne diseases were irradiated individually, all except Escherichia coli and Streptococcus faecalis showed typical linear dose-survival curves in Hartsell's broth. The minimal lethal dose (MLD) for the organisms tested ranged from 3.0 x 10(5) to 6.0 x 10(5) rad. Salmonella paratyphi B, S. wichita, S. typhi, E. coli, and S. faecalis were found to be the least sensitive to radiation. In commercially canned crabmeat the survival curves of S. typhi, S. paratyphi B, and S. wichita exhibited to varying degrees an initial linear death decline with increasing radiation doses, followed by a distinct tailing effect caused by survival of low numbers at the higher doses. The above species of Salmonella were further individually subjected to gamma-radiation in various dilutions of crabmeat. The "tailing effect" gradually disappeared, with the dose-survival curve tending to become linear as the concentration of the crabmeat decreased.     

Development of oligonucleotide primers for the specific PCR-based detection of the most frequent Enterobacteriaceae species DNA using wec gene templates

Oligonucleotide primers were designed for the PCR-based detection of the wec gene cluster involved in the biosynthetic pathway leading to the production of enterobacterial common antigen (ECA). Escherichia coli DNA was detected using wec A, wec E, and wec F gene primers. The wec A primers were specific for E. coli. The wec E and wec F primers enabled the detection of the most frequent species of the Enterobacteriaceae found in blood and urine specimens as well as in water. The sensitivity of the assay was approximately 1.2 x 102 bacteria/mL of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections. Their role in the routine testing of contamination in drinking water and food may prove to be very useful. The DNA of Enterobacteriaceae species is detected in a first step PCR, followed by specific identification of important pathogens like E. coli O157, Shigella spp., Salmonella spp., and Yersinia spp.     

Salmonella choleraesuis proteins and their relation to proteins from bacteria of heterologous sero-groups.

A diversity of proteins was identified in the material isolated from S. choleraesuis with the help of sera prepared in rabbits with this material. The sera displayed, in agar-gel diffusions, numerous superimposed precipitation lines against proteins from: Salmonellae, Shigellae and E. coli. In contrast to proteins from S. paratyphi C, sharing identical identical 'O' 'factors, the serological activity of the S. choleraesuis proteins was impaired by heating. The immunochemical analysis of the sera before and after exhaustive absorptions with heterologous proteins exhibited a stronger relation of S. choleraesuis with S. thyphimiurium and S. Newport than with S. paratyphi C. The antibodies induced against free proteins with S. paratyphi C specificity, present in the mosaic of proteins isolated from S. choleraesuis, were removed by the respective absorption without substantial modifications of the homologous precipitation. In contrast, the absorption of the serum with proteins from either S. newport or S. typhimurium removed almost all the homologous induced antibodies. The strong relations found among species belonging to different serogroups underline the non-conformity of the empirical established serofactors.     

Surveillance of bacterial pathogens associated with acute diarrheal disease in the Republic of Korea during one year, 2003

An epidemiological survey of human enterobacterial infections was conducted to determine the prevalence of enteropathogens in the Republic of Korea during one year, 2003. We tested for infectious diseases in 26,992 stool samples obtained from people who visited clinics located in six big cities and six rural provinces. From these samples, we isolated 1,291 cases of enteritis bacterial infection (4.8%). In the urban areas, 821 cases of bacterial infection (6.4%) were identified and, in the rural areas, 479 bacterial strains (3.3%) were isolated. Seasonal patterns were seen for diarrhea associated with S. aureus, E. coli and V. parahaemolyticus, while Salmonella and Shigella infections showed slight seasonal variation. We found that S. aureus and Salmonella were more frequently isolated from children and the elderly; however, the prevalence of E. coli, V. parahaemolyticus, and Shigella were similar in different age groups. Routine monitoring of these infections is considered a worthwhile means by which to elucidate their epidemiology and modes of transmission and ultimately to control them more effectively. Continuous laboratory-based surveillance for findings of enteritis bacterial infection should be emphasized in the prevention of these infections.     

Antibiotic sensitivity pattern; experience at University Hospital, Riyadh, Saudi Arabia

Results of sensitivity testing were discussed based on examination of 5192 isolates of the various bacteria isolated from clinical specimens from King Khalid University Hospital in Riyadh, Saudi Arabia. Streptococcus pyogenes and Streptococcus pneumoniae were sensitive to penicillin and erythromycin. The sensitivity pattern of Staphylococcus aureus was also predictable as they were fairly sensitive to both methicillin (98%) and erythromycin (96%). Neisseria gonorrhoeae (27%) showed a high level of resistance to penicillin. The resistance of Haemophilus influenzae to ampicillin and chloramphenicol was low. Brucella species was sensitive to tetracycline and rifampicin; resistance to streptomycin and cotrimoxazole was minimal being 1% and 6% respectively. The resistance of E. coli, Klebsiella species and Proteus species to second and third generation cephalosporins and amikacin was fairly low ranging from 1.3% to 3%. The gentamicin resistance for these organisms was also within the acceptable range (3%-10%). Gentamicin and amikacin resistance for Pseudomonas aeruginosa was low (2-8%). Salmonella typhi was sensitive to ampicillin, cotrimoxazole, and chloramphenicol. Salmonella enteritidis, Shigella species, and enteropathogenic E. coli were highly resistant to various antibiotics. Campylobacter jejuni was sensitive to gentamicin but 6% of isolates were resistant to erythromycin. Ninety six percent of Gram-negative rods except P. aeruginosa isolated from urine of patients having urinary tract infections were sensitive to amoxycillin-clavulanic acid. In addition, P. aeruginosa showed fairly low resistance to norfloxacin which is given orally to treat cystitis caused by this organism.     

Fishmeal extract bile salt lactose agar--a differential medium for enteric bacteria

Fishmeal extract bile salt lactose agar (FEBLA), a new differential medium for enteric bacteria was developed and evaluated for its ability to grow and differentiate lactose fermenters (LF) from non-lactose fermenters (NLF) in comparison with MacConkeys agar. Performance of FEBLA was at par with the latter. On FEBLA medium, the contrast between LF and NLF colonies was pronounced and Klebsiella pneumoniae produced more mucoid colonies than on MacConkeys agar (Hi Media). Unlike MacConkeys agar, a 24 h culture of K. pneumoniae cells on FEBLA were longer and thicker with abundant capsular material around the bacilli. Escherichia coli produced long and thick cells but only after 48h. No change in cell morphology was evident with regard to Salmonella typhi, S. paratyphi A, Shigella flexneri, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Citrobacter koseri and Acinetobacter baumannii. Performance of the medium was controlled using E. coli and S. flexneri. FEBLA is simple, cost effective and may be a suitable alternative in the preliminary identification of enteric bacteria.     

Thermosensitive H1 plasmids determining citrate utilization.

Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).