Pulmonary opsonins in Klebsiella pneumoniae pneumonia in rats.

Klebsiella pneumoniae was inoculated intrabronchially into rats, and bronchoalveolar lavage fluid and sera were obtained during the ensuring pneumonia. Klebsiellae recovered by lavage were not maximally coated with C3, as judged by studies with fluorescent antibody, whereas the organisms could be coated fully with C3 by a brief incubation in rat serum. The levels of C1 and C3 in lavage fluid obtained during infection were only a small fraction of the levels in the serum, and klebsiellae were not opsonized during incubation with concentrated lavage fluid. Systemic decomplementation did not affect the severity of K. pneumoniae pneumonia, as judged by the measurement of lung weight or by the numbers of klebsiellae in the lungs, but decomplemented rats had a larger number of klebsiellae in the blood at 24 h of infection than did controls. There were fewer klebsiellae in the lungs 4 h after inoculation of preopsonized organisms than after inoculation of organisms which were incubated in control (heat-inactivated) sera. These studies indicate that the concentration of complement and heat-labile opsonins within the alveoli is lower than that in the systemic circulation and is not adequate for effective opsonization of klebsiellae.     

Acetylene reduction (dinitrogen fixation) by clinical isolates of Klebsiella pneumoniae.

Freshly isolated clinical strains of Klebsiella were tested for the ability to fix dinitrogen by the acetylene reduction assay. Ability to detect this trait was markedly affected by cultural conditions. When the test was run at 37 degrees C in the presence of yeast extract (50 mg/liter), only 1.6% of the organisms were diazotrophs, whereas this temperature without yeast extract yielded 12.9% positive cultures. The optimum condition found was 28 degrees C without yeast extract (21.9% positive); therefore, search for diazotrophy in clinical strains should not be conducted at the usual incubation temperature. There was a high incidence of indole-positive strains among diazotrophs. No such correlation was noted with any other biochemical trait or antibiotic susceptibility tested. The significance of this correlation is not apparent.     

Molecular analysis of clinical isolates of ceftazidime-avibactam-resistant Klebsiella pneumoniae

ObjectivesTo analyse the strains collected during a 1-year survey of ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae, in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype.MethodsClinical KPC-producing K. pneumoniae isolates were collected from 31 patients in six different hospitals in Rome. For eight of the patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while multilocus sequence typing and core genome multilocus sequence typing were employed for molecular typing. Mutations associated with ceftazidime-avibactam resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated.ResultsMolecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26 of the 31 patients carried a mutated KPC variant, five had a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the bla_KPC-3 gene, four of which were novel mutational changes.ConclusionsDifferent mutations including single amino-acid substitutions, insertions or deletions within the bla_KPC gene were found in 26/31 ceftazidime-avibactam-resistant KPC-producing K. pneumoniae strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both ceftazidime-avibactam and carbapenems, raising concerns for the possible selection of a multidrug-resistant phenotype.     

Molecular epidemiology of clinical isolates of ampc producing Klebsiella pneumoniae

Purpose: AmpC producing K. pneumoniae have been increasingly reported from India but epidemiological studies are lacking. In the present study, molecular epidemiology of extended-spectrum AmpC beta-lactamases (ESACs) producing clinical isolates of K. pneumoniae prevalent in our hospital was studied. Methods: Fifty-one non-repeat, consecutive, clinical isolates of K. pneumoniae producing AmpC enzymes, were subjected to whole cell protein profile analysis (SDS-PAGE) and ribotyping. The antimicrobial susceptibility was determined using standard disk diffusion technique. The isolates showing decreased susceptibility to cefoxitin (<18 mm) or cefotetan (<16 mm) were subjected to modified three-dimensional test for detection of AmpC enzyme. Results: Six different types of protein profiles were observed. Ribotyping could further discriminate between the strains that were clustered by protein fingerprinting. Twelve different ribo-patterns were identified. Ribotyping was found to have a better Discriminatory Index (0.98) than that of SDS-PAGE (0.78). Of the 26 isolates that showed decreased susceptibility to cefoxitin and/or cefotetan 13 isolates were found to harbour AmpC enzyme. Conclusions: The study demonstrated the usefulness of SDS-PAGE whole cell protein profile analysis and ribotyping to identify the clonality of the ESACs isolates, the latter having a higher discriminatory power. The presence of ESACs isolates in the community as well as in hospital settings emphasizes the need for regular monitoring of antimicrobial resistance.     

Detection of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli.

Forty clinical isolates of Escherichia coli and 141 isolates of Klebsiella pneumoniae that either transferred ceftazidime resistance or showed sulbactam enhancement of oxyimino-beta-lactam susceptibility were tested by disk diffusion methodology for susceptibility to aztreonam, cefotaxime, ceftazidime, and cefoxitin. With standard 30 micrograms antibiotic disks, the fraction of these extended-spectrum beta-lactamase (ESBL)-producing isolates testing resistant by National Committee for Clinical Laboratory Standards criteria was lowest (24%) with cefotaxime disks. Forty percent of the E. coli and 29% of the K. pneumoniae isolates appeared susceptible with at least one oxyimino-beta-lactam disk. Ceftazidime and aztreonam disks were equivalent in differentiating ESBL production, and both were superior to cefotaxime disks. Over half the E. Coli and 29% of the K. pneumoniae isolates tested cefoxitin resistant. In 30 isolates, cefoxitin resistance was transmissible and due to a plasmid-mediated AmpC-type beta-lactamase. With a 5-micrograms ceftazidime disk, a breakpoint could be chosen with high sensitivity and specificity for ESBL-producing organisms. Present disk diffusion criteria underestimate the prevalence of ESBL-producing strains.     

Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey

To determine the prevalence and genotypes of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Klebsiella pneumoniae and Escherichia coli, we performed antibiotic susceptibility testing, pI determination, induction testing, transconjugation, and DNA sequencing analysis. Among the 509 isolates collected from 13 university hospitals in Korea, 39.2% produced ESBLs. ESBL-producing isolates were detected in every region in Korea. A total of 44.6% of the isolates produced both TEM- and SHV-type ESBLs, and 52% of ESBL-producing isolates transferred resistance to ceftazidime by transconjugation. The ESBLs were TEM-19, TEM-20, TEM-52, SHV-2a, SHV-12, and one new variant identified for the first time in Korea, namely, TEM-116. TEM-1 and SHV-12 were by far the most common variants. TEM-1, TEM-116, and SHV-12 showed a high prevalence in K. pneumoniae. Two isolates (E. coli SH16 and K. pneumoniae SV3) produced CMY-1-like beta-lactamases, which play a decisive role in resistance to cefoxitin and cefotetan, as well as TEM-type enzymes (TEM-20 and TEM-52, respectively). Using MIC patterns and DNA sequencing analysis, we postulated a possible evolution scheme among TEM-type beta-lactamases in Korea: from TEM-1 to TEM-19, from TEM-19 to TEM-20, and from TEM-20 to TEM-52.     

Immunization against fatal experimental Klebsiella pneumoniae pneumonia.

The ability of serospecific anti-capsular polysaccharide (CPS) antibody to prevent fatal Klebsiella pneumoniae pneumonia was evaluated in a rat lung model. Rats were immunized intramuscularly with 100 micrograms of purified serotype 2 CPS and challenged intrabronchially 14 days later with a serotype 2 strain of K. pneumoniae. Vaccination engendered high levels of serum anti-CPS antibody which afforded significant protection (P less than 0.01) against fatal pneumonia. Immunization promoted clearance of the challenge bacteria from the lungs and prevented bacteremia. Histological examination of lung tissue from infected control animals showed pronounced inflammatory cellular infiltrate in the alveolar spaces, intra- and peribronchial inflammation, and tissue necrosis. In contrast, pathological changes noted in lungs from immunized animals were restricted to infrequent intra- and peribronchial involvement.     

肺炎克雷伯临床菌株新基因组岛KpGI-2的鉴定

目的 研究肺炎克雷伯临床耐药菌株新基因组岛KpGI-2的结构和功能.方法 采用PCR方法自一株肺炎克雷伯耐药菌株HS04160扩增得到新的插入序列KpGI-2,通过测序以及生物信息学方法分析其序列结构并推测其生物学功能,采用细菌生长试验证实其生物学功能.结果 phe55 tRNA基因位点处PCR扩增得到的6.4 kb的插入序列KpGI-2,序列分析发现其GC含量为38.03%,明显低于肺炎克雷伯菌株基因组平均的GC含量(57.5%).KpGI-2中含有163 bp的重复序列,可以确定为一个新的基因组岛.KpGI-2携带有5个orf,其中orf5与沙门菌属中的Fic(fllamentation induced by cAMP)蛋白具有高度相似性.肺炎克雷伯临床菌株普遍携带有一个高度保守的fic基因以及一个相对保守的fic基因,携带有KpGI-2的临床菌株HS04160丢失了相对保守的fic基因.通过细菌生长试验分析发现orf5以及KpGI-2在cAMP诱导下对细胞生长具有调节作用,KPGI-2的功能可能与细菌生长的调节相关.结论 KpGI-2是位于肺炎克雷伯临床菌株基因组岛插入热点phe55 tRNA基因位点上的一个新基因组岛,其携带的基因与细胞生长相关.     

Aminoglycoside resistance in clinical Escherichia coli and Klebsiella pneumoniae isolates from Western Norway

Resistance to gentamicin in Escherichia coli from blood culture has shown an increase over the past decade in Norway. This study was done to investigate aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae in Western Norway. The material included 49 blood culture isolates which had shown aminoglycoside resistance collected during 2000-2009. To investigate co-resistance to alternative antibiotics and dynamics involved in aminoglycoside resistance 67 isolates (mostly from urine) exhibiting resistance to both aminoglycosides and extended spectrum beta-lactam antibiotics were also included. MIC values were obtained for amikacin, gentamicin, kanamycin, netilmicin, streptomycin and tobramycin and all isolates were screened using PCR for aac(3)-II and aac(6')-Ib, encoding aminoglycoside modifying enzymes. Resistance to ≥3 aminoglycosides was found in 92% of the isolates and 60.3% showed resistance to gentamicin, netilmicin, tobramycin and kanamycin. Amikacin resistance was low. Co-resistance to ciprofloxacin was found in 88% of the isolates with gentamicin resistance. aac(3)-IIa/c was found in 79.3% and aac(6')-Ib in 37.9% of the isolates and 28.4% harboured both genes. aac(6')-Ib-cr, possibly contributing to ciprofloxacin resistance was found mostly in extended spectrum beta-lactamase producers. The aminoglycoside resistance patterns indicate co-existence of multiple resistance mechanisms. The use of ciprofloxacin and third generation cephalosporins is likely to have contributed to the increase in aminoglycoside resistance in Norway.     

Importance of a lipopolysaccharide-containing extracellular toxic complex in infections produced by Klebsiella pneumoniae.

A Klebsiella pneumoniae serotype 2 strain was examined for its ability to produce extracellular toxic material. The organism was grown to the stationary phase in a defined medium, and the toxic material was isolated by ultrafiltration-ion-exchange chromatography on DEAE-Sephacel and gel filtration chromatography on Sepharose 4B or 2B. It was found to be comprised of 63% capsular polysaccharide, 30% lipopolysaccharide, and 7% protein and possessed a 50% lethal dose (when injected intraperitoneally into mice) of 393 +/- 45 micrograms. The toxicity appeared to be associated with the endotoxin portion of the compound, because boiling for 15 min and exposure to proteolytic enzymes had no effect on the toxicity. However, saponification destroyed the toxicity of the compound. Studies employing radial immunodiffusion examining the sera of mice infected with this organism demonstrated in vivo production of the complex at levels sufficiently high to produce death. When sublethal amounts of this complex were placed in the lungs of specific-pathogen-free mice, the lung pathology observed after 24, 48, and 72 h was similar to the damage caused by an active K. pneumoniae lobar pneumonia. These data indicate that this extracellular toxic compound produced by K. pneumoniae may be responsible for the lethality and lung tissue destruction normally associated with an active lobar pneumonia caused by this organism.     

产AmpC酶大肠埃希菌和肺炎克雷伯菌中整合子与多重耐药的研究

目的探讨整合子介导的耐药机制在产AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药中的作用.方法5株产AmpC酶大肠埃希菌和肺炎克雷伯菌分离自2002年1月-2004年5月间我院呼吸科住院的患者,采用E-test试验条进行药敏试验、电转化试验,筛选、分离耐药质粒.PCR扩增Ⅰ型整合子基因盒插入序列,分子克隆和序列分析.结果所有产酶菌株通过电转化试验可将头孢西丁耐药性传递给受体菌,5个产AmpC酶耐药质粒中,有4个检出整合酶序列,其中3个携带2种抗药性基因盒,包括氨基糖苷乙酰转移酶基因aacA4;氨基糖苷腺苷转移酶基因aadA5;二氢叶酸还原酶基因dfrA17;氯霉素外排蛋白编码基因cmL44.结论整合子介导的抗药性基因盒参与了产质粒AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药的形成,应引起高度重视.     

白细胞介素22及其受体在大鼠肺炎克雷伯菌肺炎中的表达

目的 探讨白细胞介素22(IL-22)及IL-22受体IL-10R2在大鼠肺炎克雷伯菌肺炎发病过程中表达的意义。方法 50只SPF级SD大鼠按随机数字法分为肺炎组(n=25)和对照组(n=25)。肺炎组通过气管内注射肺炎克雷伯菌建立大鼠肺炎克雷伯菌肺炎模型,对照组则滴入等体积无菌生理盐水。造模后4h、1、2、3、4d通过观察大鼠的一般情况、肺组织大体和病理切片、肺组织细菌菌落计数对肺炎模型进行评价。采用实时荧光定量PCR和ELISA分别检测肺炎组和对照组大鼠造模后4h、1、2、3、4d肺组织IL-10R2 mRNA、外周血IL-22的表达水平。应用直线相关方法分析菌落计数与IL-22及其受体的相关性。结果肺炎组大鼠造模后4h出现活动度下降、聚集成团、竖毛现象;大体肺组织明显充血水肿,显微镜下肺泡结构破坏、肺泡腔及间质显著的充血及水肿、显著的中性粒细胞浸润。肺炎组大鼠肺组织均可培养出细菌,造模后4h细菌菌落计数最高[(0.94±0.02)×105 CFU/g],之后逐渐降低,至4d时降至最低[(0.07±0.00)×105 CFU/g],证实肺炎模型制备成功。与对照组比较,肺炎组IL- 10R2mRNA的表达量于造模后4h开始升高,于第3天达峰值（12.95±0.65比0.31±0.31,P＜0.05）,其后开始下降。肺炎组外周血中IL-22均低于同时间点对照组。肺炎组外周血中IL-22自造模后第3天较4h开始明显升高且随时间的延长逐渐升高。肺炎组中IL-10R2 mRNA、外周血IL-22的表达水平均与同时间点肺组织菌落计数呈负相关（r=-0.64,P=0.01;r=-0.59,P=0.00）。结论 IL-10R2在肺炎克雷伯菌肺炎中高表达,而外周血中IL-22可能由于消耗导致降低,但可随肺炎的好转回升。IL-22是肺炎克雷伯茵肺炎早期的炎性反应因子,在体内具有清除细菌的作用。     

Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates

A multilocus sequence typing (MLST) scheme was developed for Klebsiella pneumoniae. Sequences of seven housekeeping genes were obtained for 67 K. pneumoniae strains, including 19 ceftazidime- and ciprofloxacin-resistant isolates. Forty distinct allelic profiles were identified. MLST data were validated against ribotyping and showed high (96%) discriminatory power. The MLST approach provides unambiguous data useful for the epidemiology of K. pneumoniae isolates.     

Seroepidemiology of clinical isolates of Klebsiella in Connecticut.

The distribution of capsular serotypes of 200 clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca from four Connecticut hospitals was determined. Serotyping was done by an indirect fluorescent-antibody technique. Hospitals included three community hospitals from the Hartford area and one university hospital in New Haven. During the test period, epidemiological surveillance did not detect any nosocomial epidemic involving Klebsiella species. Ninety-two percent of the isolates were typable. Of the 72 possible serotypes, 62 were represented among these strains. Forty-two percent of the typable strains were distributed among 10 serotypes. The predominant serotypes were types 31, 22, and 18 representing 19% of the typable strains (8, 6, and 5%, respectively). No one particular serotype was associated exclusively with a specific site of infection.     

Relationship between adhesion to intestinal Caco-2 cells and multidrug resistance in Klebsiella pneumoniae clinical isolates.

Klebsiella pneumoniae is an opportunistic gram-negative pathogen involved in outbreaks of nosocomial infections in intensive care units. Strains are resistant to multiple antibiotics, and 15 to 30% of them are also resistant to the broad-spectrum cephalosporins by the production of R plasmid-encoded extended-spectrum beta-lactamases. Because the gastrointestinal tracts of patients have been shown to be the reservoir for nosocomial strains of K. pneumoniae, we looked for a correlation between antibiotic resistance and adhesion of K. pneumoniae strains to intestinal cells. We investigated adhesion to the human intestinal epithelial Caco-2 cell line of 61 clinical K. pneumoniae strains isolated in hospitals in Clermont-Ferrand, France. None of the strains tested expressed the previously described adhesive factors CF29K and KPF-28. Adhesive properties were found for 42.6% of the strains tested (26 strains). Just 7.7% (2 strains) of the 26 strains producing only the chromosomally encoded SHV-1 beta-lactamase adhered to the Caco-2 cell line, whereas 68.5% (24 strains) of the 35 strains producing a plasmid-encoded beta-lactamase were adherent. All the adherent strains, and even the two strains producing only the SHV-1 enzyme, harbored at least one self-transmissible R plasmid. At variance for CAZ-1/TEM-5 or CAZ-5/SHV-4 beta-lactamase-producing K. pneumoniae strains, curing and mating experiments demonstrated that the self-transmissible R plasmids encoding the TEM-1, CTX-1/TEM-3, CAZ-2/TEM-8, CAZ-6/TEM-24, or CAZ-7/TEM-16 beta-lactamase were not involved in the adhesion of K. pneumoniae strains to intestinal epithelial cells. Nevertheless, there was an association of multiple antibiotic resistance, including resistance to extended-spectrum cephalosporins, and adhesive properties in K. pneumoniae clinical isolates.     

Evaluation of differential gene expression in susceptible and resistant clinical isolates of Klebsiella pneumoniae by DNA microarray analysis

DNA microarray technology was used to evaluate differential gene expression in a susceptible Klebsiella pneumoniae isolate and a resistant clinical derivative. Nineteen genes were up-regulated in the resistant isolate when compared with the susceptible isolate. An ABC transporter-related gene, ycjV, was strongly over-expressed, suggesting the existence of a novel active efflux mechanism. Approximately half of the up-regulated genes coded for ribosomal proteins, or proteins involved in tRNA metabolism. Among 33 downregulated genes, almost one-third were related to nitrogen metabolism. A possible role of fitness in the development of antimicrobial resistance is suggested.     

Occurrence of the Yersinia high-pathogenicity island and iron uptake systems in clinical isolates of Klebsiella pneumoniae

The ability to acquire iron is crucial to bacteria during an infection. Thirty-four strains of Klebsiella pneumoniae isolated from clinical specimens were examined for the use of various strategies to obtain iron. The isolates employed several iron uptake mechanisms, including production of enterobactin (100%) and aerobactin (50%). Few isolates (18%) produced yersiniabactin, a siderophore encoded by the Yersinia high-pathogenicity island (HPI) despite genetic diversity of the HPI. Majority of the isolates used human transferrin (74%), lactoferrin (97%), hemoglobin (74%), and hemoglobin-haptoglobin complex (56%) as a sole source of iron. Multiple iron uptake systems may be of benefit to the bacteria during infection.