Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.     

PCR-based identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the agent of rhinoscleroma

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma.     

Incidence of Klebsiella species in surface waters and their expression of virulence factors.

To investigate the occurrence of different Klebsiella spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species being Klebsiella pneumoniae. A comparison of these isolates to a group of 207 clinical K. pneumoniae isolates demonstrated that water isolates of K. pneumoniae, unlike those of K. oxytoca and K. planticola, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.     

头孢西丁耐药肺炎克雷伯菌AmpC基因和ESBLs检测及耐药性分析

目的了解深圳地区头孢西丁耐药肺炎克雷伯菌头孢菌素酶(AmpC酶)基因型分布、产超广谱β-内酰胺酶(ESBLs)的情况及其耐药特点。方法收集深圳地区三家大型综合医院临床标本分离对头孢西丁耐药的肺炎克雷伯菌73株。用碱裂解法提取菌株的质粒,采用多重PCR扩增AmpC基因,应用DNA测序确定其基因型。并对所有菌株进行ESBLs表型确证试验;用K-B法对其进行药物敏感试验。结果 48株(65.8%)AmpC基因扩增阳性,经DNA测序显示,其中46株为DHA-1型,1株为CMY-2型,1株同时产DHA-1和CMY-2型;73株肺炎克雷伯菌中49株ESBLs阳性,其中36株AmpC基因和ESBLs均为阳性。AmpC和(或)ESBLs阳性菌株对多数药物的耐药率高于AmpC和ESBLs均阴性者。结论本地区头孢西丁耐药肺炎克雷伯菌质粒AmpC酶检出率高,基因型主要为DHA-1,同时产AmpC酶和ESBLs菌株较常见。     

Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide.

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.     

PCR鉴定肺炎克雷伯菌的强毒性血清型

目的:了解肺炎克雷伯菌强毒性血清型K1、K2、K54和K57型菌株在我国重庆、北京、深圳三地的分布及流行趋势。方法:采用PCR对310株肺炎克雷伯菌临床分离株进行血清型K1、K2、K54和K57检测。结果:310株菌中,K1、K2、K54和K57血清型分别占14.2%、9.4%、6.5%和4.2%;来自呼吸系统标本分离株中的K1血清型菌株在4种检测的强毒血清型中占首位,为呼吸系统总数的17.4%。结论:310株肺炎克雷伯菌的4种强毒性血清型中,K1血清型菌株所占比例高,较为流行。     

Identification of species and capsular types of Klebsiella clinical isolates, with special reference to Klebsiella planticola

In the 77 reference strains for Klebsiella K types, there are 17 strains (22.1%) of Klebsiella planticola, 6 strains (7.8%) of Klebsiella oxytoca, 1 strain (1.3%) of Klebsiella terrigena, and 53 strains (68.8%) of Klebsiella pneumoniae. The species K. planticola, which was originally isolated from botanical and aquatic environments and hence thus named, was also identified at high incidence (81 strains, 18.5%) among the 439 recent clinical isolates of Klebsiella species. Among these K. planticola strains of hospital origin, 52 (64%) were isolated from sputum, 17 (21%) from urine, and the remaining 12 (15%) from other sources. The capsular types of these isolates were determined by the gel precipitation reaction. Seventy of 81 K. planticola isolates (86.4%) were typable by antisera to Klebsiella reference strains for K types and the K types of the clinical isolates distributed to 35 kinds of K types. The proportion of typable strains among clinical isolates of K. planticola was very similar to those in K. pneumoniae (87.5%) and K. oxytoca (86.0%).     

Incidence of two virulence factors (aerobactin and mucoid phenotype) among 190 clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamase

Because outbreaks of multiple-resistant Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases were recently observed in French hospitals, the presence of virulence factors was examined for (i) phenotype by bioassay for aerobactin production and by culture for the mucoid phenotype, and (ii) genotype using intragenic probes of respectively 2-kb BglII and 235-bp BamHI-BglII fragments and dot-blotting among 190 unreplicated K. pneumoniae clinical isolates issued from 25 French hospitals and producing different types of extended-spectrum beta-lactamases (TEM-related enzymes: TEM-3, TEM-4, CAZ-1, CAZ-2, TEM-8, or SHV-related enzymes: SHV-2, SHV-3, SHV-4). Only 3.7% and 7% of K. pneumoniae isolates produced aerobactin and mucoid phenotypes respectively, unrelated to type of beta-lactamase. Only 2% had both factors. No discordance was reported according to the detection method tested. The low prevalence of such virulence factors seems to indicate they were not involved in dissemination of nosocomial K. pneumoniae isolates producing an extended-spectrum beta-lactamase.     

Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes

A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.     

Do Neutrophils Play a Role in Establishing Liver Abscesses and Distant Metastases Caused by Klebsiella pneumoniae?

Serotype K1 Klebsiella pneumoniae is a major cause of liver abscesses and endophthalmitis. This study was designed to identify the role of neutrophils in the development of distant metastatic complications that were caused by serotype K1 K. pneumoniae. An in vitro cellular model was used to assess serum resistance and neutrophil-mediated killing. BALB/c mice were injected with neutrophils containing phagocytosed K. pneumoniae. Serotype K1 K. pneumoniae was significantly more resistant to serum killing, neutrophil-mediated phagocytosis and intra-cellular killing than non-K1 isolates (p<0.01). Electron microscopic examination had similar findings as in the bioassay findings. Intraperitoneal injection of neutrophils containing phagocytosed serotype K1 K. pneumoniae led to abscess formation in multiple sites including the subcutaneous tissue, lung, and liver, whereas no abscess formation was observed in mice injected with non-K1 isolates. The resistance of serotype K1 K. pneumoniae to complement- and neutrophil-mediated intracellular killing results in the dissemination of K. pneumoniae via the bloodstream. Escape from neutrophil intracellular killing may contribute to the dissemination and establishment of distant metastases. Thus, neutrophils play a role as a vehicle for helping K. pneumoniae and contributing to the establishment of liver abscess and distant metastatic complications.     

Virulence factors (aerobactin and mucoid phenotype) in Klebsiella pneumoniae and Escherichia coli blood culture isolates

Abstract We examined the presence of two virulence factors in 241 blood isolates of Klebsiella pneumoniae from patients hospitalized during 1989 and 1990 in 7 French hospitals, and 125 blood isolates of Escherichia coli from one hospital. Aerobactin was scored phenotypically and genotypically with an intragenic DNA probe of 2 kb. The mucoid phenotype was assessed by culture on trypticase soy agar and by genotypic analysis (intragenic DNA probe of 235 bp). Only 6% K. pneumoniae isolates were aerobactin-positive with no significant variation according to geographical location while 20% of K. pneumoniae isolates displayed the mucoid phenotype, with a significant variation according to hospital. Aerobactin was always associated with the mucoid phenotype. The frequency of aerobactin production but not mucoid phenotype (14%) was higher among E. coli isolates (48%). They harbored two types of large plasmids. Intraperitoneal injection into mice of 10³ cfu of K. pneumoniae producing both virulence factors demonstrated that capsular serotype K2 was the more virulent K23 and K28.     

High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess

To better understand the role of capsular polysaccharide (CPS) K1 or K2 in Klebsiella pneumoniae liver abscess as well as the development of metastasis to eye, neutrophil phagocytosis of 70 CPS isolates including K1 (n = 23)/K2 (n = 10), non-K1/K2 (n = 37) was evaluated by flow cytometry, fluorescence imaging, and electron microscopy. K1/K2 isolates were significantly more resistant to phagocytosis (P < 0.0001) than non-K1/K2 isolates and displayed increased resistance to intracellular killing. Although mucoid phenotype (M-type) K1/K2 isolates were significantly more resistant to phagocytosis (P = 0.0029) than M-type non-K1/K2, no significant difference in the phagocytosis rate was observed between K1/K2 isolates with M-type and non-M-type (P = 0.0924). Mucoidy is an associated factor that was predominant in K1/K2 isolates, but which itself is not an independent influence on phagocytic resistance. The K1/K2 CPS proved significantly more resistant to phagocytosis than non-K1/K2 CPS in liver abscess isolates (P < 0.0001) and non-abscess isolates (P = 0.0001), suggesting that K1/K2 isolates were generally more virulent in both liver abscess and in non-liver abscess conditions. These findings indicate that resistance of CPS K1 or K2 K. pneumoniae to phagocytosis and intracellular killing presumably contributes to their high prevalence in liver abscess and uniquely in endophthalmitis.     

Differentiation of Aerobacter-Klebsiella isolated from sugarcane

Three hundred and eighty-four isolates were obtained in the completed test portion of the most probable number determinations of coliforms in sugarcane sources. Of these isolates, 88% were of the (- - + +) indole, methyl red, Voges-Proskauer, citrate (IMViC) type and were identified as Aerobacter aerogenes according to the protocol of the American Public Health Association (1). Employing 359 of these cultures, a comparative biochemical, serological, and pathogenicity study was carried out with Klebsiella pneumoniae CDC no. 2211-66 type 9. More than 86% of the organisms tested gave biochemical reactions typical of K. pneumoniae. Of the other isolates, 2% were Enterobacter aerogenes, and the remaining 12% were identified as atypical, nonmotile IMViC types. Comparable agglutination titers were also observed between A. aerogenes and the CDC strain of K. pneumoniae when several randomly selected sugarcane strains were reacted with prepared K. pneumoniae whole cell antiserum. Neither the K. pneumoniae reference organism nor selected sugarcane isolates displayed pathogenicity for mice. On the basis of all the analyses performed, it was suggested that such organisms be classified as K. pneumoniae.     

Role of microbiological surveillance in the choice of antibacterial therapy for intestinal Klebsiella infection in infants

Data on the frequency of intestinal infections caused by Klebsiella spp. in infants at the age of 1 month to 1 year and antibiotic resistance of K. pneumoniae and K. oxytoca isolates from newborns and infants aged 1 month to 1 year are presented. The frequency of the Klebsiella isolates from the newborns and infants at the age of 1 month to 1 year with acute intestinal infections amounted to 17.7 +/- 1.4 and 42.5 +/- 1.4 per cent, respectively. The majority of the clinical strains had a multiple resistance to 7-9 drugs. The overwhelming majority of the strains were sensitive to gentamicin, monomycin, kanamycin, neomycin and polymyxin B.     

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。     

长沙某医院产质粒AmpC酶型肺炎克雷伯菌的多重PCR检测与分析

为了了解湖南长沙某医院临床分离的肺炎克雷伯菌中质粒介导AmpC β-内酰胺酶的产生情况及其基因型,收集了该医院2008年3月至2010年10月临床分离的多重耐药肺炎克雷伯菌104株,用头孢西丁纸片扩散法对这些菌株进行表型初筛,用多重PCR确定ampC耐药基因型;结果发现其中有19株对头孢西丁纸片不敏感,疑为产AmpC酶菌株;再经多重PCR扩增,有12株菌分别在约400 bp（11株）和约350 bp（1株）出现了阳性条带,特异性PCR证明此12株菌分别携带了DHA型（11株）和ACC型（1株）ampC耐药基因;产质粒介导AmpC酶肺炎克雷伯菌的分离率为11.5%（12/104）。该医院产质粒介导AmpC酶肺炎克雷伯菌的分离率较高,应对其检测与监测给予足够重视,以指导临床合理选用抗菌药物。     

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.     

Incidence of Extended-Spectrum β-Lactamases and Characterization of Integrons in Extended-Spectrum β-Lactamase-producing Klebsiella pneumoniae Isolated in Shantou, China

This study is concerned with the level of antibiotic resistance of extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae, isolated in Shantou, China, and its mechanism. Seventy- four non-repetitive clinical isolates of K. pneumoniae producing ESBLs were isolated over a period of 2 years. Antibiotic susceptibility, carried out by Epsilometer test, showed that most of the isolates were multiresistant. Polymerase chain reaction showed that, among the several types of β-lactamases, SHV was the most prevalent, TEM was the second most prevalent, and CTX-M was the least prevalent. Sixty-nine isolates were positive for integrase gene IntI1, but no IntI2 or IntI3 genes were found. The variable region of class 1 integrons were amplified and further identified by sequencing. Thirteen different gene cassettes and 11 different cassette combinations were detected. Dfr and aadA cassettes were predominant and cassette combinations dfrA12, orfF and aadA2 were most frequently found. No gene cassettes encoding ESBLs were found. Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.     

Occurrence of selected loci of the cps region for capsule K1 or K2 synthesis in epidemic strains of Klebsiella pneumoniae isolated from infants in Poland

Number of 60 epidemic strains of K. pneumoniae and 15 isolated occasionally from stool samples were tested for presence of 4 and 11 selected loci of the cps cluster of K1 and K2. Following open reading frames (ORFs): 1, 2, 3, 4, 7, 9, 10, 14, 15 (gnd) of cps K2 strain Chedid and genes magA, gmd, wzc, wca of cps K1 strain DTS were searched by PCR in tested and reference strains O1:K1 A5054 and O1:K2 B5055. The ORFs 1 to 3 and ORF15 were detected in both the reference and epidemic strains as well as in the greatest majority of the occasional isolates. Thus, such ORFs were found K. pneumoniae cps common domains, while tested ORFs 4 to 14 were observed in strain B5055 and 11 epidemic isolates from patients of the same hospital ward. Only exception was a single strain O3 occasionally isolated from faeces. The tested genes of cps K1 were detected only in strain A5054 and in two O3 occasional isolates from faeces. Interestingly, these genes as well ORFs 4 to 14 were detected together in the appropriate reference and tested strains with only two exceptions. Therefore, the cps sector occupied by ORFs 4 to 14 was found as group-specific domain. The occurrence ratio of cps K2 group-specific loci among epidemic strains from infants was 18%, while the K1 group-specific loci were absent.     

重症监护病房产ESBLs菌的基因分型

目的了解深圳市人民医院重症监护病房分离菌超广谱β-内酰胺酶（ESBLs）的检出率及其基因型分布情况。方法收集来自重症监护病房大肠埃希菌和肺炎克雷伯菌分离株48株,采用CLSI推荐的表型确证方法筛选出ESBLs株,并利用PCR及DNA测序法分析产酶菌株的ESBL基因型。结果（1）48分离株菌中共检出产ESBLs菌24株,阳性率为50.0%。（2）产酶菌中93.8%（15/16）的大肠埃希菌和87.5%（7/8）的肺炎克雷伯菌分别检出CTX-M基因;其中72.7%（16/22）为CTX-M-14。6株肺炎克雷伯菌检出SHV基因,其中3株为SHV-11型,另3株为SHV-12型,6株含SHV基因的肺炎克雷伯菌中5株合并CTX-M基因。而所有大肠埃希菌株均未检出SHV基因。所有产酶菌中,分别有10株大肠埃希菌和2株肺炎克雷伯菌检出TEM-1基因,其中1株大肠埃希菌只检出TEM-1基因,未检出SHV型或CTX-M型基因。结论重症监护病房分离菌ESBLs检出率高,以CTX-M-14为主要基因型。