Inhibition ofPseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics

The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of quinolones (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin), aminoglycosides (amikacin, gentamicin, netilmicin, streptomycin, tobramycin), β-lactams (aztreonam, ceftazidime, imipenem, ticarcilin) and macrolides (erythromycin, roxitromycin) on the excretion of alginate by aP. aeruginosa strain were studied. Both β-lactam and macrolide antibiotics were found ineffective at the concentrations tested, except erythromycin and imipenem at 1/4 MIC. Aminoglycosides at a concentration of 1/4 MIC reduced most effectively the excretion of alginate. Quinolones were also effective at this sub-MIC; 1/16 MIC was ineffective with all antibiotics or stimulated the production of alginate.     

Providencia isolates carrying bla (PER-1) and bla (VIM-2) genes: biofilm-forming capacity and biofilm inhibitory concentrations for carbapenem antibiotics

Multidrug-resistant clinical isolates of Providentia carrying bla _PER-1 and bla _VIM-2 were evaluated for the abilities to form biofilm and high biofilm forming capacity was demonstrated in them. Minimum biofilm inhibitory concentrations (MBICs), minimum biofilm eradication concentrations (MBECs), and minimum inhibitory concentrations (MICs) for imipenem and meropenem were also determined. In all tested strains, the MBICs were higher than the MICs for both drugs. Interestingly, the MBICs and the MBEC₅₀ for meropenem were lower than those for imipenem in the isolates producing high amounts of biofilm, suggesting that meropenem is superior to imipenem in the growth inhibition and eradication of biofilm forming Providentia strains.     

Postantibiotic effects of imipenem and enoxacin againstS. typhimurium andS. enteritidis and the influence on their surface hydrophobicity

The influence of the postantibiotic effect (PAE) and the postantibiotic sub-MICs effect (PA SME) of imipenem and enoxacin on the surface hydrophobicity ofS. typhimurium andS. enteritidis strains were studied by evaluating Congo red binding and the aggregation in molar solutions of ammonium sulfate (SAT). A PAE was induced by 2× and 4× MIC of antibiotics tested for 0.5 h. Suprainhibitory concentrations of imipenem againstS. typhimurium induced a short PAE (0.3–0.6 h) compared toS. enteritidis (6.0–9.7 h). Suprasubinhibitory concentrations of imipenem did not allow a regrowth ofS. enteritidis. Similar results were also found for enoxacin. Evaluation of surface hydrophobic properties of the salmonellas after affecting both PAEs and PA SMEs has shown that imipenem at concentrations 4×MIC and 4×MIC+0.3×MIC partially influenced the hydrophobicity ofS. typhimurium. S. enteritidis was more susceptible toward both antibiotics tested.     

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Combined Porin Loss and Extended Spectrum β-Lactamase Production is Associated with an Increasing Imipenem Minimal Inhibitory Concentration in Clinical <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Strains

For this study, 150 clinical isolates of Klebsiella pneumoniae were collected from one hospital in Beijing, China, and assayed for minimal inhibitory concentration (MIC) of imipenem. To elucidate the mechanisms responsible for imipenem MIC variation among extended-spectrum β-lactamase (ESBL)-positive and -negative K. pneumoniae strains, a variety of β-lactamase genes (bla _TEM, bla _CTX-M, bla _SHV, and bla _OXA) were screened by polymerase chain reaction (PCR). The outer membrane profile and expression of related genes (ompK35 and ompK36) then were analyzed and evaluated, respectively. None of the tested isolates were clinically resistant to imipenem, but the range of MICs among ESBL-positive and -negative strains was significantly different. Deficiency in the expression of outer membrane proteins (OmpK35,36) was observed in some of both ESBL-positive(17.6%) and -negative strains (10.9%), but only the ESBL-positive strains depressed by the expression of ompK35/36 had an increased MIC of imipenem (≥0.5 mg/l). These results confirmed that the combination of SHV-1, CTX-M-3, CTX-M-14, TEM-1, or OXA-11 production and reduced expression of ompK35/36 may not result in clinical resistance to imipenem but does correlate with increasing imipenem MIC.     

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> CL180

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.     

革兰氏阴性杆菌氟喹诺酮耐药株的实验研究

对８２株临床分离的氟喹诺酮药物（Ｆｌｕｏｒｏｑｕｉｎｏｌｏｎｅｓ,ＦＱＮＳ）耐药菌进行了多种抗生素敏感试验。结果显示８２株耐药菌对５种药物耐药率分别为：诺氟沙星９７．５６％、依诺沙星９６．３４％、罗美沙星９５．１２％、氧氟沙星９２．６８％、环丙沙星８７．８％。３２株菌对ＦＱＮＳ存在较普遍的交叉耐药现象,对氨苄青霉素、庆大霉素、丁胺卡那霉素及头孢菌素类的多重耐药率也较高。８２株菌中有４３株菌携带质粒（５２．４４％）。质粒消除及转化实验结果表明：质粒与ＦＱＮＳ耐药性无关。     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.     

Lipolytic activity of dermatophytes

Lipolytic activity of dermatophytes was tested by the method generally used forCandida lipolytica. Most of the freshly isolated strains ofMicrosporum canis, M. gypseum, Epidermophyton floccosum andTrichophyton mentagrophytes gave positive reactions, whereas, only few strains ofT. schoenleini, T. violaceum, T. megnini, T. rubrum andT. tonsurans yielded such reactions. Among subcultured strains, it was found a decreased lipolytic activity inMicrosporum species and an increased lipolytic activity inEpidermophyton and someTrichophyton species.Part of this work was performed at Serviço de Doenças Infecto-Contagiosas (Secção de Dermatologia), Hospital de Curry Cabral - Lisboa.     

Extracellular lipolytic activity in Phoma glomerata

Several properties of the lipolytic activity exhibited by the conidial fungus Phoma glomerata were studied. Lipolytic activity in an aqueous buffer medium was measured on triacylglycerol, phosphoglyceride and cholesterol ester under different experimental conditions. The effect of storage temperature on the stability of the hydrolytic activity, and optimal conditions of temperature and time of maximal activity were determined. The optimal conditions for maximal lipolytic activity were found to be 40–50 °C and 1 h. The activity released to the medium by 1 mg cells for 1 h at 40 °C was stated as the enzyme released unit (ERU). The protein fraction of MW > 50 kDa obtained by ultrafiltration of the medium, was active on the three substrates assayed, and it showed a non-specific hydrolytic activity on both the 1- and 2-acyl esters either in the neutral glyceride or in the phosphoglyceride. A protein of M _r approx. 75 kDa was the only one that showed esterase activity. The crude medium, stored at –15 °C, maintained its initial hydrolytic activity on triacylglycerol for at least 42 days, though when it was kept for 10 days at 4 °C, the activity fell to 50%. Kinetic parameters using substrates such as triolein (TO), dipalmitoyl phosphatidylcholine (DPPC) and cholesteryl oleate (ChoO), were comparatively evaluated. The activity of the enzyme in the hydrolysis of TO showed the highest values, whereas the maximal specific activities were less when the enzyme was assayed against DPPC and ChoO.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Technological characteristics of yeast strains and their potential as starter adjuncts in Greek-style black olive fermentation

The technological properties of Debaryomyces hansenii (15 strains) and Torulaspora delbrueckii (32 strains) isolated from Greek-style black olives under conditions typical for black olive fermentation were studied. Furthermore, the killer character of the strains was assessed as well as their antimicrobial action against food-borne pathogens. All strains could grow at 15°C and low pH (2.5), whereas the majority of the strains were able to grow at 10% (w/v) NaCl, assimilated d-galacturonic acid, and showed lipolytic activity. Only 33% of D. hansenii and 9% of T. delbrueckii strains could hydrolyse 1% (w/v) oleuropein. A large majority of the strains tolerated 0.3% (w/v) bile salts, which in correlation with acid resistance indicates probiotic potential. Cross-reactions between culture filtrates of D. hansenii and T. delbrueckii and 56 yeast strains isolated during spontaneous Greek-style black olive fermentation were conducted. Focusing on their lytic activity, 17 mycogenic strains were selected. Culture filtrates of the mycogenic strains inhibited strains of L. monocytogenes, B. cereus and S. typhimurium. The active substance was heat resistant (stable after heating at 100°C for 10 min) as well as stable over a pH range from 4.0 to 6.5. The possible inhibition of undesirable yeast contaminants and food-borne pathogens in situ on fermented olives as well as the probiotic potential of strains used as starter adjuncts would contribute to the improvement of quality of the fermented product.     

Phenolic acids reduce the genotoxicity of acridine orange and ofloxacin inSalmonella typhimurium

Naturally occurring plant phenolics,p-coumaric acid (PA), caffeic acid (CA), ferulic acid (FA) and gentisic acid (GA) (25–100 nmol/L) had protective effects on acridine orange (AO; 216 μmol/L)- and ofloxacin (3 μmol/L)-induced genotoxicity inSalmonella typhimurium. FA, GA and CA exhibited a significant concentration-dependent protective effect against the genotoxicity of AO and ofloxacin, with the exception of PA, which at all concentrations tested abolished the AO and ofloxacin genotoxicity. UV spectrophotometric measurements showed the interaction of PA, FA, GA and CA with AO but not with ofloxacin; this interaction is obviously responsible for the reduction of AO-inducedS. typhimurium mutagenicity. In the case of ofloxacin the antimutagenic effect of PA, FA, GA and CA is assumed to be a result of their ability to scavenge reactive oxygen species (ROS) produced by ofloxacin.     

In Vitro Activities of Fourteen Antimicrobial Agents Against Drug Susceptible and Resistant Clinical Isolates of Mycobacterium tuberculosis and Comparative Intracellular Activities Against the Virulent H37Rv Strain in Human Macrophages

Minimal inhibitory concentrations (MICs) of 14 first and second-line antituberculous drugs against drug-susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis (including the multiple drug-resistant or MDR-TB isolates), as well as the type strain H37Rv, were determined radiometrically by the Bactec 460-TB methodols. MICs (μg/ml) of all the fourteen drugs were within an extremely narrow range in case of susceptible strains; isoniazid (0.02–0.04), rifampin (0.2–0.4), ethambutol and streptomycin (0.5–2.0), ethionamide (0.25–0.5), D-cycloserine (25–75), capreomycin (1–2), kanamycin (2–4), amikacin (0.5–1.0), clofazimine (0.1–0.4), ofloxacin (0.5–1.0), ciprofloxacin (0.25–1.0), and sparfloxacin (0.1–0.4). The activity of second-line drugs remained unaltered against MDR-TB isolates resistant to routine first-line drugs. With peak serum level concentrations (Cmax), the intracellular killing of the virulent H37Rv strain was studied in detail in cultured human macrophages. Based on an decreasing order of bactericidal activity, our results showed the following spectrum of intracellular drug action: among the first-line drugs, rifampin > ethionamide = isoniazid > ethambutol > streptomycin > D-cycloserine; among second-line drugs, clofazimine = amikacin > kanamycin = capreomycin; among fluoroquinolones, sparfloxacin > ofloxacin > ciprofloxacin. On the other hand, contrary to atypical mycobacteria, the macrolide drug clarithromycin was inactive against both extracellular and intracellular M. tuberculosis. Received: 23 January 1996 / Accepted: 5 April 1996     

Influence onEnterobacter cloacae metabolism, cell-surface hydrophobicity and motility of suprainhibitory concentrations of carbapenems

The impact of postantibiotic effect (PAE) of carbapenems (imipenem, meropenem) on the metabolism (biosynthesis of macromolecules, respiration), cell-surface hydrophobicity and motility of a clinical isolate ofEnterobacter cloacae was examined. The metabolism was evaluated after 16 h and after 1 d of cultivation using 2× and 4× minimum inhibitory concentrations (MIC) of both antibiotics for the induction of PAE. Imipenem at 4×MIC did not induce PAE. After a 16-h cultivation (in the postantibiotic phase of both carbapenems), inhibition of nucleosynthesis and protein synthesis was found; after a 1-d cultivation, during regrowth stimulation of mainly¹⁴C-leucine incorporation was found. The presence of the exogeneous intermediates of citrate cycle,viz. 2-oxoglutarate, increased the respiratory activity of the cells. The cell-surface hydrophobicity (evaluated by three methods—bacterial adhesion to hydrocarbon, nitrocellulose-filter test and salt-aggregation test) decreased after PAE of both carbapenems; meropenem was more effective. Motility (an important virulence factor) was inhibited in the postantibiotic phase of both carbapenems; the 4×MIC caused a higher inhibition.     

In vitro Activity of Gatifloxacin Against 238 Strains of Anaerobic Bacteria

The activity of gatifloxacin, a new 8-methoxy-fluoroquinolone, was tested against 208 pulmonary pathogens and against an additional 30 isolates of the Bacteroides fragilis group. Pulmonary isolates were from patients with documented anaerobic pleuropulmonary infections and were obtained by appropriate sampling methods. MICs were determined using the NCCLS-approved Wadsworth brucella laked blood agar method and compared to those of clindamycin, imipenem, metronidazole and trovafloxacin. Breakpoints used to define susceptible and [resistant] categories were (in μg/ml): Clindamycin-2, imipenem-4, metronidazole 8 and trovafloxacin. No breakpoint has been defined for gatifloxacin. Gatifloxacin inhibited 99% of all anaerobes tested at 4 μg/ml and 97% of all strains at 2 μg/ml. One strain of B. fragilis was resistant to gatifloxacin at 4 μg/ml; all strains of other B. fragilis group species were susceptible. One strain of Peptostreptococcus sp. was resistant to both gatifloxacin and trovafloxacin (MIC >4 μg/ml). All other strains were susceptible to all agents at ≤μg/ml. All of the non-sporeforming Gram-positive rods were susceptible to gatifloxacin at ≤μg/ml (three strains had an MIC of 4 μg/ml). Trovafloxacin had MICs of 4 μg/ml for two strains, and an MIC of 8 μg/ml for one strain. Five percent of B. fragilis, 21% of other B. fragilis group species and 20% of Clostridium species (other than C. difficile, C. perfringens or C. ramosum) were resistant to clindamycin. No imipenem resistant isolates were found in this study. Gatifloxacin appears to have excellentin vitro activity against pulmonary isolates of anaerobes and very good activity against strains of the B. fragilis group.     

Characteristics of clinical<Emphasis Type="Italic">Acinetobacter</Emphasis> spp. strains

Resistance to 13 antimicrobial agents, resistance to the bactericidal activity of human serum, hydrophobic properties, lipolytic activity and production of histamine were determined in a total of 50 clinicalAcinetobacter spp. strains (A. baumannii, A. lwoffii, A. calcoaceticus, A. haemolyticus). None of the tested isolates showed resistance to meropenem and none ofA. lwoffii, A. calcoaceticus andA. haemolyticus strains were resistant to amikacin. Forty-six strains (92 %) manifested resistance to ampicillin, 90 % to cefuroxime, 68 % to ciprofloxacin, 58 % to piperacillin, gentamicin and cotrimaxazole, 50 % to cefotaxime, 44 % to amikacin, 42 % to ceftazidime, 38 % to piperacillin/tazobactam, 24 % to netilmicin and 16 % to ampicillin/sulbactam. In particular,A. baumannii andA. calcoaceticus strains showed considerable antibiotic resistance. Thirty-one isolates (62 %) showed serum resistance; intermediate sensitivity was found in 19 isolates (38 %). The majority of the strains (72 %) demonstrated a strongly hydrophobic character; 16 % of isolates exhibited moderate hydrophobic properties. All strains showed lipolytic activity; production of histamine was detected in 14 of 43 strains examined.     

Diphenolases from <Emphasis Type="Italic">Anoxybacillus kestanbolensis</Emphasis> strains K1 and K4 <Superscript>T</Superscript>

Summary Diphenolases from Anoxybacillus kestanbolensis strains K1 and K4^T, highly active against 4-methylcatechol were characterized in terms of pH- and temperature-optima, pH- and temperature-stability, kinetic parameters, and inhibition/activation behaviour towards some general polyphenol oxidase (PPO) inhibitors and metal ions. The temperature-activity optima, for Anoxybacillus kestanbolensis K1 and K4^T catecholases in the presence of 4-methylcatechol, were 80 and 70 °C, respectively. Although catecholase from A. kestanbolensis K4^T lost no activity after a period of 1 h incubation at its optimum temperature, the enzyme pH from K1 was stimulated by keeping at 80 °C. Both of the enzymes possessed pH optima at 9.5, and the pH-stability profiles showed that cathecholases from both preparations retained their activities at alkaline pH values. Both A. kestanbolensis K1 and K4^T catecholase activities were totally inhibited by addition of 0.01 mM sodium metabisulphite, ascorbic acid and l-cysteine. 1 mM Mn²⁺ increased the activities of A. kestanbolensis K1 and K4^T catecholases by 6.4- and 5.3-fold, respectively. These results indicate that both A. kestanbolensis K1 and K4^T strains possess thermo- and alkalostable catecholases.     

Isolation and identification of killer yeasts from Agave sap (aguamiel) and pulque

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic beverages such as beer and wine; therefore, killer or killer-resistant strains have been sought for use in alcoholic fermentations. In the current paper a total of 16 strains belonging to six species were isolated. From two samples of Agave sap (aguamiel) the following yeast strains were isolated: Candida lusitaneae (1), Kluyveromyces marxianus var. bulgaricus (2), and Saccharomyces cerevisiae (capensis) (1). Additionally, in seven samples of pulque (the fermented product), the species C. valida (six strains), S. cerevisiae (chevalieri) (4), S. cerevisiae (capensis) (1), and K. marxianus var. lactis (1) were found. The killer strains were C. valida and K. marxianus var. lactis from pulque and K. marxianus var. bulgaricus from aguamiel. One strain of S. cerevisiae (chevalieri) isolated from pulque which did not show killer activity was, on the other hand, resistant to other killer strains and it had a remarkable ethanol tolerance, suggesting that this strain could be used for alcohol production.