Detection of inducible β-lactamase by an agar dilution technique

Abstract An agar dilution technique for the detection of inducible β-lactamase-mediated resistance to the newer cephalosporins is described. Cefoxitin (16 μg/ml) was incorporated in agar plates together with cefamandole (8 μg/ml) or cefotaxime (8 μg/ml). The susceptibility of 35 strains of Enterobacter cloacae to these combinations was compared with their susceptibility to the individual antimicrobial agents. Of 31 strains which could be evaluated, 18 (58%) produced an inducible β-lactamase which inactivated cefamandole, while 10 (32%) produced an inducible cefotaxime-inactivating enzyme. The technique has the advantages of being 24 h faster than the alternative disc-diffusion induction test, and of being suitable for testing large numbers of strains simultaneously.     

Co-existence of β-lactamase and penicillin acylase in bacteria; detection and quantitative determination of enzyme activities

Twenty-six bacteria were examined for the presence of penicillin acylase and β-lactamase. A copper reducing assay, which was sensitive in the analytical range 2–20 μg/ml, was used for determination of penicilloates and a fluorescamine assay was used to determine 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate. Seventeen bacteria contained β-lactamases, six contained penicillin acylases and four contained both enzymes. Two bacteria contained a Type 1 penicillin acylase and four bacteria contained a Type II enzyme. No ampicillin acylases were detected. All β-lactamases were constitutive enzymes in those organisms where both enzymes co-existed. Bacillus subtilis and B. cereus produced inducible and extracellular β-lactamases. Acinetobacter calcoaceticus ATCC 21288 produced a constitutive β-lactamase which was detected extracellularly.     

LXA-1: a new plasmid-mediated β-lactamase giving low-level resistance

Abstract LXA-1, a novel plasmid-mediated β-lactamase, was observed in clinical isolates of Klebsiella oxytoca, Klebsiella pneumoniae, Citrobacter freundii and Enterobacter cloacae . All the strains additionally produced TEM-1 β-lactamase. LXA-1 had an M _r of 24 000 and a pI of 6.7. It hydrolysed benzyl-penicillin, ampicillin, carbenicillin and first generation cephalosporins, but not methicillin, oxacillin or cefotaxime. Clavulanate and cloxacillin were inhibitors. Studies of one of the E. cloacae isolates showed that LXA-1 was encoded by a 41-MDa IncFII plasmid distinct from that encoding TEM-1 enzyme in the strain. Transconjugants which acquired LXA-1 production, but not TEM-1, exhibited only low-level resistance to substrate β-lactams.     

Combinations of Clavulanic Acid and other β-lactam Antibiotics against Strains of Pseudomonas aeruginosa, Serratia marcescens and other Bacteria

Combinations of clavulanic acid, a new β-lactamase inhibitor, with five cephalosporins and one cephamycin were tested against cell-free β-lactamases obtained from Serratia marcescens, Pseudomonas aeruginosa and an Enterobacter strain, 265A. Cefotaxime was the most resistant antibiotic and cephalothin the most sensitive antibiotic to β-lactamases. Low concentrations of clavulanic acid gave some protection against the Serratia and Pseudomonas enzymes. The most active source of β-lactamase was the 265A strain, against which only cefotaxime was highly resistant. Clavulanic acid had only a slight inhibitory effect on this enzyme, which was confirmed by an agar method, and potentiated slightly the activity of cephalothin and cefoxitin against two β-lactamase producing strains of Staphylococcus aureus. Lysis by cephalothin of one strain of S. marcescens was potentiated in the presence of clavulanic acid.     

产AmpC酶阴沟肠杆菌的基因分析及其耐药性

探讨昆明地区阴沟肠杆菌的耐药性及与结构基因ampC和调节基因ampD的相关性。通过K-B法检测阴沟肠杆菌的药敏情况,头孢西丁三维试验检测AmpC酶,PCR法扩增ampC和ampD基因。结果显示74株阴沟肠杆菌经头孢西丁三维试验检测,产AmpC酶的有17株,检出率为22.3%,而且产酶菌株抗生素敏感率低于非产酶菌株。ampC基因扩增阳性率为89.2%(66/74);64株ampD基因阳性率为86.5%(64/74)。实验证实昆明地区产酶阴沟肠杆菌耐药状况严重,与结构基因ampC和调节基因ampD密切相关。     

Cloning of a beta-lactam resistance determinant of Enterobacter cloacae affecting outer membrane proteins of Enterobacteriaceae

Abstract In this paper we describe the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that causes β-lactam resistance in both Escherichia coli HB101 and the parental strain E. cloacae 2249-1.
The increase in minimum inhibitory concentration (MIC) of the β-lactam antibiotics studied was not the result of enhanced β-lactamase production, but of a decrease in the concentration of the pore proteins OmpF and OmpC in E. coli and of a 37-kDa membrane protein in E. cloacae . The results obtained thus far indicate that we have cloned a gene encoding a 20 kDa polypeptide that is involved in the regulation of outer membrane protein synthesis.     

Thiosulfate Oxidation and Tetrathionate Reduction by Intact Cells of Marine Pseudomonad Strain 16B

Levels of thiosulfate-oxidizing enzyme (TSO) and tetrathionate reductase (TTR) were measured in washed cell suspensions of a heterotrophic marine thiosulfate-oxidizing bacterium, strain 16B. TSO activity remained virtually constant in aerobically and anaerobically grown cells and was unaffected by the presence or absence of thiosulfate and tetrathionate in the growth medium. TTR was also present in cells grown aerobically and anaerobically, but its activity was threefold greater in cells cultured in media containing tetrathionate or thiosulfate. Tetrathionate appears to be the inducer of increased TTR activity in both aerobically and anaerobically grown cells. TTR (constitutive or induced) and TSO have different pH and temperature optima. Both TTR activities were unaffected by 10 mM KCN, which reversed oxygen inhibition of tetrathionate reduction. TSO was partially inhibited by 5 μM KCN and completely inhibited by 90 μM KCN. These findings and results of experiments to determine the influence of several inorganic electron donors and acceptors on TSO and TTR activities suggest that constitutive TSO and TTR represent reverse activities of the same enzyme, whereas inducible TTR is a separate enzyme used by strain 16B only for anaerobic respiration of tetrathionate. The bacterium appears well adapted to growth in environments characterized by low oxygen tension, dilute organic carbon concentrations, and the presence of a variety of reduced, inorganic sulfur compounds.     

Isolation and partial purification of a carbapenem-hydrolysing metallo-β-lactamase from Pseudomonas cepacia

Abstract A metallo-β-lactamase has been isolated from a clinical strain of Pseudomonas cepecia and partially purified using Cibacron blue F3GA coupled agarose. The resulting preparation showed a single band of β-lactamase activity (p I 8.45) after analytical isoelectric focusing. The enzyme was particularly effective in the hydrolysis of imipenem. Meropenem, biapenem, cephaloridine, ceftazidine, benzylpenicillin, ampicillin and carbenicillin were also hydrolysed, although at a lower rate. An unusual inhibition profile was noted. Inhibition by the metal ion chelators ethylenediaminetetraacetic acid and o -phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst > 90% inhibition was attainable with 0.1 mM concentrations of tazobactam and clavulanic acid. A study of 8 other clinical isolates showed the enzyme to be present and inducible by imipenen in each case. This enzyme was assigned PCM-I ( Pseudomonas cepacia metalloenzyme I).     

229株阴沟肠杆菌的标本分布及耐药性分析

目的 了解阴沟肠杆菌在医院感染的标本分布和耐药情况,为临床合理选择和应用抗生素提供依据.方法 采用VITEK 60全自动微生物分析仪和配套的GNI、GNS-143、GNI-448,对229株阴沟肠杆菌进行分离鉴定和药敏试验,药敏结果使用WHONET 5.5软件进行分析.结果 从2007年1月至2011年9月共分离到阴沟肠杆菌229株,59.0％来自于呼吸道标本,其次是尿液占13.5％,再次为创面分泌物/脓液占12.7％.阴沟肠杆菌对美洛培南、亚胺培南和头孢哌酮/舒巴坦的耐药率分别为0.0％、0.4％和2.5％,对氨苄西林、头孢唑啉、头孢西丁的耐药率分别为98.7％、96.9％、97.5％.结论 阴沟肠杆菌耐药机制复杂,对抗生素具有多重耐药性,临床应合理使用抗菌药物,以减少阴沟肠杆菌耐药性的产生和在院内扩散.     

Chromate-resistance in a chromate-reducing strain of Enterobacter cloacae

Resistance to toxic hexavalent chromium (chromate: CrO4(2)) in Enterobacter cloacae strain HO1, isolated from an activated sludge sample, was investigated under aerobic and anaerobic conditions. Decreased uptake of 51CrO4(2-) in E. cloacae strain HO1 was observed under aerobic conditions, when compared with a standard laboratory E. cloacae strain (IAM 1624). Under anaerobic conditions E. cloacae strain HO1 was able to reduce hexavalent chromium to the less toxic trivalent form. When E. clocacae strain HO1 was grown with nitrate anaerobically, the cells were observed to lose simultaneously their chromate-reducing ability and chromate-resistance under anaerobic conditions.     

Biochemical and Clinical Characteristics and Antibiotic Susceptibility of Atypical Enterobacter cloacae

The characteristics of an atypical group of the family Enterobacteriaceae resembling Enterobacter cloacae were studied. The urinary tract was the most common source of these organisms, and most strains represented infections of secondary clinical significance. In contrast to typical Enterobacter strains, the atypical strains were highly susceptible to the cephalosporins; otherwise, there was a high degree of susceptibility to five other antibiotics and resistance to ampicillin except in very high concentration.     

Membrane-associated chromate reductase activity from Enterobacter cloacae.

Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.     

老年患者阴沟肠杆菌感染的临床特点及耐药性分析

目的了解我院老年患者阴沟肠杆菌感染的临床分布及耐药性变迁,为临床合理用药提供参考依据。方法采用回顾性分析方法,统计临床数据并分析我院2011年9月至2016年4月期间老年患者感染标本中分离出的阴沟肠杆菌的感染现状及耐药性。结果共检出149株阴沟肠杆菌,主要分离于痰液、全血和尿液中,分别占31.54%、24.16%和18.12%。在科室分布中,阴沟肠杆菌感染主要来源于普通外科、重症监护病房和呼吸内科,分别占26.84%、14.10%和14.10%。药敏结果显示阴沟肠杆菌对美罗培南、亚胺培南和阿米卡星具有较好的抗菌活性,敏感率分别为100.00%、96.64%和95.97%,而对阿莫西林/克拉维酸、氨苄西林、头孢替坦、头孢西丁和头孢唑啉的耐药率分别为96.36%、96.36%、97.67%、100.00%和100.00%。结论阴沟肠杆菌易引起呼吸道、泌尿道以及伤口的感染,且其耐药现象较为严重,应加强耐药性的监测,根据药敏结果合理选用抗菌药物,以控制医院感染。     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Integration of exogenous DNA into the genome of Azotobacter vinelandii

The soil bacterium Azotobacter vinelandii was genetically transformed by chromosomal integration to ampicillin and/or tetracycline resistance using restriction endonuclease-linearized plasmids. Polyacrylamide gel electrophoresis of protein extracts from three independently isolated ampicillin resistant transformants showed the presence of a 28 Kd band which is the approximate size of the ampicillin resistance gene product (i.e., -lactamase). Moreover, with nitrocefin, a chromogenic cephalosporin, as a substrate, it was shown that all of the ampicillin resistant transformants produced functional -lactamase. DNA hybridization showed that the chromosomal DNA from transformed cells contained plasmid DNA sequences at discrete sites. Growth experiments indicated that stable A. vinelandii transformants that carry functional integrated DNA were physiologically impaired.     

Evaluation of a commercial β-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial β-glucuronidase (β-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be β-GUR positive. Thirty-one clinical isolates of Shigella sonnei , 10 of Enterobacter cloacae , eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme β-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the β-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were β-GUR negative and one C. freundii was β-GUR positive. Escherichia coli was the only species positive for both β-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. β-GUR positive Enterobacter strains have not previously been described.     

Inhibition of Aerobacter Cephalosporin β-Lactamase by Penicillins

Cephalosporinase (beta-lactamase) was obtained from cell washings of Aerobacter (Enterobacter) cloacae as a highly active preparation. An alkalimetric method was used to determine the enzyme activity and to estimate its inhibition by 6-amino-penicillanic acid derivatives. Their order of decreasing inhibitory effect was as follows: cloxacillin, oxacillin, methicillin, ampicillin, and penicillin G. We found that 2 to 3 ng of cloxacillin per ml was sufficient to decrease the enzyme activity by 50% in the presence of 400 mug of cephalosporin C per ml. Cloxacillin exerted a potentiating effect on the inhibition of the E. cloacae organisms by cephalosporin C.     

Serotypes and pyocin types of Pseudomonas aeruginosa isolated from natural waters

The susceptibility of strains of Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Proteus mirabilis and Escheriehia coli to six aminoglycosides was tested in media of different osmolarity and ionic content. We observed that increasing osmolarity decreased susceptibility of these Gram-negative bacteria to all antibiotics used. On the other hand, raising of ionic strength increased the susceptibility to tobramycin, neomycin and gentarnicin in all bacteria tested.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.