In vitro antimicrobial activities of new quinolone antibiotics against Mycoplasma pneumoniae]

The antimicrobial activities against Mycoplasma pneumoniae of new quinolones (temafloxacin, ofloxacin, ciprofloxacin, enoxacin, and norfloxacin) and of tetracyclines and macrolides as controls were compared. Among new quinolones, temafloxacin, ofloxacin, and ciprofloxacin were more active than enoxacin and norfloxacin against fifty strains of Mycoplasma pneumoniae, giving MIC50 and MIC90 significantly lower than those of the latter two, by the agar-dilution method. The three more active antibiotics in the above assay were then determined for MICs and MBCs by the broth-dilution method. The MICs of every antibiotic except erythromycin determined by both the methods were very similar each other. The MICs of erythromycin determined by the broth-dilution method were ten-times higher than those determined by the agar-dilution method. Temafloxacin and ofloxacin gave MBCs only about four-times higher than MICs, whereas ciprofloxacin, minocycline, erythromycin and josamycin gave MBCs as much as 15 to 1,000-times higher than MICs. From the MICs and MBCs determined by the two assay methods, it is apparent that temafloxacin and ofloxacin, and to a less extent ciprofloxacin, have more potent mycoplasmacidal activities than do macrolides and tetracyclines.     

肺炎克雷伯杆菌对氟喹诺酮耐药的机制研究

目的研究肺炎克雷伯杆菌对氟喹诺酮类药物（FQNs）的耐药机制。方法筛选临床分离的对环丙沙星耐药的肺炎克雷伯杆菌共10株,采用微量肉汤稀释法检测菌株对5种氟喹诺酮类药物的MIC值;采用PCR方法检测菌株染色体和质粒携带的喹诺酮耐药基因（gyrA基因、parC基因和qnr基因）并测序;质粒接合试验验证qnr基因的转移性。结果 10株肺炎克雷伯杆菌对5种氟喹诺酮类药物均产生耐药性。扩增产物经测序发现10株肺炎克雷伯杆菌染色体的gyrA基因和parC基因均有突变;有2株菌株（K79和K107）携带qnrA基因,这2株菌的接合菌对喹诺酮抗菌药的MIC值上升了5～30倍;未检测到qnrB阳性的菌株。结论 gyrA和parC基因突变是肺炎克雷菌对氟喹诺酮类产生耐药机制的主要原因,质粒上qnrA基因的存在,也是产生喹诺酮耐药的一个重要因素。     

Pathogenic potential of environmental Klebsiella pneumoniae isolates

Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. K. pneumoniae infections can occur at nearly any body site; however, urinary tract infections and infections of the respiratory tract predominate. Infections are frequently preceded by gastrointestinal colonization, and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. In contrast to many other bacterial pathogens, K. pneumoniae is ubiquitous in nature. Several studies have described Klebsiella isolates of environmental origin to be nearly identical to clinical isolates with respect to several phenotypic properties. However, the pathogenic potential of environmental K. pneumoniae isolates is essentially unknown. We have evaluated the virulence of K. pneumoniae strains of environmental and clinical origin directly in animal models, i.e. in urinary tract infection and intestinal colonization models. Furthermore, the ability to adhere to and invade human epithelial cell lines was examined. Although strain-to-strain differences were observed in the individual infection models, overall, strains of environmental origin were found to be as virulent as strains of clinical origin. The ubiquity of K. pneumoniae in nature and the general ability of K. pneumoniae strains to infect susceptible hosts might explain the high frequency of opportunistic infections caused by this species.     

Transformation of the antibacterial agent norfloxacin by environmental mycobacteria

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight Mycobacterium sp. cultures in a sorbitol-yeast extract medium were dosed with 100 microg ml(-1) of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 microg ml(-1). Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. N-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight Mycobacterium strains. N-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by Mycobacterium sp. strain PYR100 and Mycobacterium gilvum PYR-GCK. The MICs of N-nitrosonorfloxacin and N-acetylnorfloxacin were 2- to 38- and 4- to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although N-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.     

Comparative characteristics of the adhesive properties of microorganisms isolated from the urine of children with chronic obstructive pyelonephritis

The adhesive properties of 215 cultures, including 215 Escherichia coli strains, 43 Klebsiella pneumoniae strains and 60 Pseudomonas aeruginosa strains isolated from the urine of 124 children with chronic obstructive pyelonephritis were studied in the direct hemagglutination test simultaneously with those of 30 E. coli strains and 20 K. pneumoniae strains isolated from the feces of 50 healthy children, as well as 60 P. aeruginosa strains isolated from children with parenteral infections of other localization. E. coli and K. pneumoniae strains isolated from the urine of children with chronic obstructive pyelonephritis were found to have D-mannose-resistant hemagglutinins (68% and 37.2%) and a combination of mannose-sensitive and mannose-resistant adhesins (44.6% and 13.3% respectively). P. aeruginosa strains isolated from the urine of urological patients in the postoperative period showed the presence of mannose-resistant hemagglutinins to a greater extent (76.6%) than those isolated from children with parenteral infections of other localization (45%).     

新生儿医院感染产超广谱β-内酰胺酶细菌流行状况及耐药性分析

目的了解新生儿医院感染中产超广谱β-内酰胺酶（ESBLs）细菌流行状况及耐药性,为预防和控制感染提供依据。方法对2011年1月至2013年12月间新生儿医院感染病原菌分布及耐药性进行回顾性分析;用VITEK-2 Compact微生物鉴定系统鉴定菌种和药敏试验。结果共检出病原菌192株,105株为肺炎克雷伯菌和大肠埃希菌,占54.7%;检出产ESBLs菌58株,全部来自肺炎克雷伯菌和大肠埃希菌;产ESBLs菌对青霉素类、头孢菌素类、单内酰环类抗菌药物高度耐药,耐药率〉80.0%,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦耐药率较低,耐药率〈20.0%,未检测到亚胺培南和美罗培南耐药株。结论产ESBLs肺炎克雷伯菌和大肠埃希菌是新生儿医院感染中主要的病原菌,且对常用抗菌药物耐药率较高,临床应加强病原菌的耐药性监测,合理使用抗菌药物。     

In vitro activity of three different antimicrobial agents against ESBL producing Escherichia coli and Klebsiella pneumoniae blood isolates

Extended spectrum beta-lactamases (ESBLs) usually associated with multiple drug resistance, including beta-lactam and non-beta-lactam antibiotics. This resistance can cause Limitation in the choice of drugs appropriate for using in clinical practice, especially in life-threatening infections. In this study we aimed to investigate in vitro activity of meropenem, ciprofloxacine and amikacin against ESBL-producing and non-producing blood isolates of Escherichia coli and Klebsiella pneumoniae strains. Fifty-eight E. coli (21 ESBL-producing, 37 non-ESBL producing) and 99 K. pneumoniae (54 ESBL-producing, 45 non-ESBL producing) strains were included in the study. The presence of ESBL was investigated by double disk synergy test and E-test methods. Antibiotic susceptibility test was done by microdilution method according to NCCLS guideline. In vitro susceptibilities of ESBL producing E. coli and K. pneumoniae strains were found as 100% for meropenem, 33.3% and 25.9% for ciprofloxacine, 94.5% and 83.3% for amikacin. It was observed that; meropenem was equally active agent in both ESBL-producing and non-producing strains, and its activity was not affected by ESBL production. Whereas amikacin activity was minimally affected and ciprofloxacine activity was markedly decreased by ESBL production. In conclusion, meropenem seems to be better choice of antibiotic should be used for ESBL positive life-threatening infections, because of remaining highest activity.     

Hydrolytic and haemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

Hydrolytic enzymes and haemolysins are important extracellular substances produced by many bacteria. We investigated 57 K. pneumoniae strains and 40 K. oxytoca strains isolated from clinical materials. We estimated the ability to produce: proteases hydrolyzing milk powder, caseinase, gelatinase, elastase, lecithinase, lipases, DNase and haemolysins on human, sheep and horse erythrocytes on TSA medium with or without 5% Egg Yolk. We detected that K. oxytoca strains produced proteases hydrolyzing milk powder (37.5%), caseinase (15.0%) and gelatinase (17.5%) more frequently than K. pneumoniae strains (respectively 21.0%, 5.3%, 8.9%). None of the analysed Klebsiella spp. strains produced elastase. Only K. pneumoniae strains produced lecithinase (5.3%). Lipases hydrolyzing Tween were produced from 3.5% (for Tween 60 and 80) to 7.0% (for Tween 20). Among K. oxytoca strains only one (2.5%) hydrolyzing Tween 20. DNase was produced by 38.6% of K. pneumoniae strains and by 27.5% K. oxytoca strains. Haemolytic properties on human erythrocytes were detected in 5.3% K. pneumoniae strains on TSA medium and 29,8% on medium with Egg Yolk. In K. oxytoca strains haemolytic properties on human erythrocytes were detected only on medium with Egg Yolk (12.5%). Haemolytic properties on sheep erythrocytes were detected respectively in 21.0% and 22.8% K. pneumoniae strains and in 7.5% K. oxytoca strains on each medium. Haemolytic properties on horse erythrocytes were detected respectively in 33.4% and 52.6% K. pneumoniae strains and in 15.0% and 20.0% K. oxytoca strains.     

肺炎克雷伯菌基因组可移动遗传元件的研究进展

肺炎克雷伯菌是目前临床上最主要的耐药致病菌之一,对人类健康造成了很大威胁.近年来,细菌耐药成为治疗肺炎克雷伯菌感染的主要难题,尤其是高毒力、高耐药性肺炎克雷伯菌的出现对临床工作造成了巨大挑战,而研究表明其耐药基因和毒力基因主要由可移动遗传元件携带而传播.因此,为了更好地认识及防控肺炎克雷伯菌感染,本文对肺炎克雷伯菌基因...     

Enterotoxigenic capacity of strains of Klebsiella and Enterobacter genera isolated in acute intestinal diseases in children

234 strains, including 104 K. pneumoniae strains, 28 K. oxytoxica strains, 64 E. cloacae strains and 40 E. aerogenes strains, have been isolated from the intestine of 266 children with diarrhea, aged up to 1 year, and studied for enterotoxigenicity. By the coagglutination test, made with G. Kronvall's staphylococcal reagent prepared with the use of antiserum to Escherichia coli LT-enterotoxin, and the biological assay on suckling mice enterotoxigenic activity has been revealed in 119 strains, including 48 K. pneumoniae strains (12.6%), 33 E. cloacae strains (27.4%) and 23 E. aerogenes strains (19.7%). The strains producing only LT-enterotoxins, only ST-enterotoxins, and both LT- and ST-enterotoxins have been found. The determination of the enterotoxigenic activity of the clinical isolates of opportunistic enterobacteria makes it possible to improve the etiological interpretation of acute intestinal infections.     

Ammonia enhanced dark respiration in Chlorella vulgaris is related to collapse of a transmembrane pH gradient

Abstract We obtained, by different methods, isogenic lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen) mutants from Klebsiella pneumoniae strains able to induce experimental infections (cytitis and pyelonephritis) in rats. We compared the induction of experimental infections in rats by wild-type strains and the lipopolysaccharide and capsular polysaccharide mutants. The high-molecular mass lipopolysaccharide of K. pneumoniae is clearly implicated in the infection process of the rat urinary tract, whilst the capsular polysaccharide seems not to be involved to the same extent.     

重症监护病房肺炎克雷伯菌ESBLs检测及耐药性分析

目的了解江山市人民医院重症监护病房肺炎克雷伯菌产超广谱β-内酰胺酶（ESBLs）的情况及其耐药性分析。方法收集2006年1月至2011年12月该院重症监护病房临床分离的肺炎克雷伯菌418株,采用VITEK-32全自动细菌鉴定仪;药物敏感试验采用K-B纸片扩散法,用双纸片协同试验进行ESBLs检测。采用聚合酶链式反应（PCR）扩增检测细菌产ESBLs的基因分型。结果 418株肺炎克雷伯菌主要来源于痰液标本和中段尿标本等,其中产ESBLs的肺炎克雷伯菌有111株。重症监护病房肺炎克雷伯菌年龄分布以71～80岁年龄组最高,而产ESBLs菌株则以51～60岁年龄组为最高,可达43.1%。产ESBLs菌株对一～三代头孢菌素、氯霉素、大多数氟喹诺酮类抗菌药物耐药率一直很高;对碳青霉烯类抗菌药物总体上有较高的敏感性,但耐药菌也在有所出现。PCR扩增检测ESBLs基因型,该院中CTX-M、SHV、TEM等均占较高比例,提示上述三种耐药基因型在本地区均有分布。结论医院重症监护病房临床分离的肺炎克雷伯菌产ESBLs的比例较高,尤其是泛耐药菌株的不断出现,给临床治疗带来了巨大难题,应引起高度重视,以预防重症监护病房感染的发生和暴发流行。     

Hemagglutinating and adhesive capacities of Klebsiella and Enterobacter strains

The authors analyze the data of studies on the hemagglutinating and adhesive capacity of 290 cultures, including 118 K. pneumoniae strains and 64 E. cloacae strains isolated from sick children, as well as 59 K. pneumoniae strains and 49 E. cloacae strains isolated from healthy children. The hemagglutinating properties of the strains were determined in the hemagglutination test with fresh, formalin- and tannin-treated red blood cells, the adhesive properties were studied by light microscopy. Among K. pneumoniae and E. cloacae strains isolated in acute intestinal infections, mannose-sensitive hemagglutination and pronounced adhesive activity were prevalent in most cases. Poorly adhesive and nonadhesive strains were characteristic of K. pneumoniae and E. cloacae cultures isolated from healthy children. The strains isolated from sick and healthy children differed only by the prevalence of adhesive cultures.     

Transformation of the Antibacterial Agent Norfloxacin by Environmental Mycobacteria

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight Mycobacterium sp. cultures in a sorbitol-yeast extract medium were dosed with 100 μg ml⁻¹ of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 μg ml⁻¹. Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. N-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight Mycobacterium strains. N-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by Mycobacterium sp. strain PYR100 and Mycobacterium gilvum PYR-GCK. The MICs of N-nitrosonorfloxacin and N-acetylnorfloxacin were 2- to 38- and 4- to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although N-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.     

Problems of prevalance and resistance of nosocomial Klebsiella pneumonia in hospitals of Russia]

Prevalence of Klebsiella pneumoniae among gramnegative pathogens of nosocomial infections in intensive care units of 33 hospitals of 22 towns in Russia was investigated. Antibiotic susceptibility and extended-spectrum beta-lactamase production were tested in 420 nosocomial K. pneumoniae isolates. Carbapenems (imipenem, meropenem and ertapenem) showed the highest activity. Extended-spectrum beta-lactamase production based on the phenotyping methods was revealed in 342 (81.4%) isolates. The maximum activity against the K. pneumoniae isolates producing extended-spectrum beta-lactamase was observed in imipenem and meropenem (no unsusceptible strains were isolated). 3.2% of the isolates was not susceptible to ertapenem. Differences in the activity of cefoperazone/sulbactam, amikacin, ciprofloxacin and levofloxacin against the extended-spectrum beta-lactamase producing isolates in various hospitals were recorded.     

A carbapenem-resistant Klebsiella pneumoniae isolate harboring KPC-1 from Italy

Carbapenem-resistant gram-negative pathogens represent an emerging threat to the management of hospital-acquired infections. Although the isolation of carbapenem-resistant enterobacteriaceae remains unusual, the frequency of carbapenemases producing Klebsiella pneumoniae is increasing in different geographic regions: the majority of isolates has been collected in the USA, but recently KPC-producing K. pneumoniae were reported from China, Israel, Greece, France, Norway and Sweden. We report a KPC 1-producing K. pneumoniae isolate from Italy. This datum enlarges the geographical area where the KPC-producing K. pneumoniae strains are diffuse.     

产ESBLs肺炎克雷伯菌AmpC酶的检测及耐药性分析

目的了解深圳市人民医院产ESBLs肺炎克雷伯菌中产AmpC酶的情况及其耐药性。方法收集产ESBLs肺炎克雷伯菌临床株126株,应用Tris-EDTA纸片法检测AmpC酶。用琼脂稀释法测定菌株对11种抗生素的最低抑菌浓度（MIC）。结果126株ESBLs阳性的肺炎克雷伯菌中12株检出AmpC酶,检出率为9.5%。AmpC阳性菌株对头孢西丁、头孢他啶、氨曲南、氨苄西林/舒巴坦和阿莫西林/克拉维酸的耐药率达100%,对阿米卡星和哌拉西林/他唑巴坦的耐药率分别为83.3%和33.3%,其中头孢西丁、头孢他啶、氨曲南、阿莫西林/克拉维酸、阿米卡星和哌拉西林/他唑巴坦的耐药率显著高于AmpC阴性株（P〈0.05）。结论深圳市人民医院产ESBLs肺炎克雷伯菌中检出AmpC酶阳性株,其耐药性强于单产ESBLs菌株。     

革兰阴性杆菌ESBLs和AmpC酶的检测及耐药分析

目的检测引起医院感染的革兰阴性杆菌携带产ESBLs和去阻遏AmpC酶状况,探讨各菌对临床常用抗生素的主要耐药机制及耐药性,为临床制定合理使用抗生素策略提供依据。方法采用全自动微生物分析仪（VITEK-32）做细菌鉴定和药敏试验,用纸片扩散确证法检测超广谱β-内酰胺酶（ESBLs）,用三维法检测高水平表达染色体编码的AmpC酶。结果158株革兰阴性杆菌ESBLs检出率为26．6％,主要菌为大肠埃希菌（45．2％）、肺炎克雷伯菌（42．9％）、阴沟肠杆菌（11．9％）。AmpC酶检出率为10．1％,主要为鲍曼不动杆菌（43．8％）、阴沟肠杆菌（25％）;上述产酶细菌均对青霉素和一、二、三代头孢菌素、磺胺类、喹诺酮类、氨基糖苷类耐药,对亚胺培南敏感。结论革兰阴性杆菌耐药机制主要是产超广谱β-内酰胺酶和AmpC酶,这些产酶菌株均出现多重耐药。     

连续12年产超广谱β-内酰胺酶肺炎克雷伯菌感染分布及耐药性分析

目的了解产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌（KPN）的临床分布及耐药性,为临床合理选用抗菌药物提供依据。方法用常规方法,结合法国梅里埃公司的自动化细菌鉴定分析仪分离鉴定细菌;用K-B纸片扩散法进行药敏试验;用双纸片协同筛选法和酶抑制剂增强纸片法检测ESBLs。对中国医科大学附属盛京医院1999年1月至2010年12月期间临床分离的产ESBLs和非产ESBLs KPN菌株的临床分布及其耐药趋势进行回顾性分析。结果 KPN的ESBLs检出率分别从1999年的19.82%上升至2010年的49.34%;KPN主要分离于痰占52.4%,其次是血液占17.74%,主要来源于外科病区和新生儿病区;产ESBLs菌和非产ESBLs菌对阿米卡星、左氧氟沙星等非β-内酰胺酶类抗菌药物的耐药率均显著增高,差异具有统计学意义（P〈0.01）,对亚胺培南和美罗培南都保持高度敏感,耐药率均低于2%。结论 12年间ESBLs检出率总体呈上升趋势,产ESBLs肺炎克雷伯菌表达对包括非β-内酰胺类在内的抗菌药物多重耐药,对碳青霉烯类抗菌药物仍保持高度敏感性。