Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay.

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum ß-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla (TEM), bla (SHV), and bla (CTX-M), and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla (CTX-M15), bla (SHV-28), and bla (TEM-1) positive by PCR. Ten out of 52 were ST16 and tested positive for bla (CTX-M15), bla (SHV-1), and bla (TEM-1). Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for bla (CTX-M15), bla (SHV-28), and bla (TEM-1), and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing bla (CTX-M15) and bla (SHV-28) constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.

Screening for vancomycin-resistant enterococci with Xpert® vanA/vanB: diagnostic accuracy and impact on infection control decision making.

Vancomycin-resistant enterococci (VRE) are increasingly important nosocomial pathogens and screening for colonization status is a mainstay in infection control. We implemented PCR-based screening during vanA-positive Enterococcus faecium outbreaks in four university hospitals in Copenhagen, Denmark. Xpert®vanA/vanB was performed directly on rectal swabs and the vanA PCR result was used to guide infection control measures. Concurrently, all samples were selectively cultured including an overnight enrichment step. Diagnostic accuracy was calculated as well as turnaround time and the impact of the earlier available PCR results on infection control decision making. In all, 1110 samples were analysed. The vanA PCR positivity rate was 13.8% and culture positivity rate was 15.2%. The diagnostic accuracy of the vanA part of the assay was high with a sensitivity of 87.1%, a specificity of 99.7%, and positive and negative predictive values of 98.0% and 97.7%, respectively. The vanB PCR had a considerably lower specificity of 77.6% and a positive predictive value of 0.4%. In 1067 (96.1%) samples, PCR results were reported within 1 day, whereas median culture turnaround time was 3 days. The saving of time to available results corresponded to 141 saved isolation days and 292 saved transmission risk days. False-negative or false-positive PCR results led to six additional transmission risk days and 13 additional isolation days, respectively. The vanA PCR had high diagnostic accuracy and the prompt availability of results gave a considerable benefit for infection control decision making.

Immune responses in Indonesian thin tail and Merino sheep during a primary infection with Fasciola gigantica: lack of a specific IgG2 antibody response is associated with increased resistance to infection in Indonesian sheep.

After a primary infection with Fasciola gigantica, the immune responses in a resistant (Indonesian thin tail) and a susceptible (Merino) breed of sheep were analysed. The number of adult flukes recovered from the livers of the Indonesian thin tail sheep were significantly lower than those found in the Merino animals. On days 8, 14 and 25 p.i., Indonesian thin tail sheep exhibited a significantly higher eosinophilia than Merino sheep, whereas neutrophilia was significantly elevated in the Indonesian thin tail sheep on days 36 and 48 p.i. Serum from both sheep breeds demonstrated IgM, IgG1 and IgE responses to F. gigantica. In contrast, the Indonesian thin tail sheep produced significantly lower levels of IgG2 antibodies relative to the high level detected in Merino sheep. The IgE response was biphasic in both sheep breeds with the first response detected by day 14 and the second response developing from days 30 to 60 p.i. Western blotting showed that a similar profile of adult fluke antigens was recognised by IgG1 and IgE antibodies in both the Indonesian thin tail and Merino sheep. The IgE response was directed to a major antigen at about 92 kDa. We postulate that IgG2 could act as a blocking antibody for protective effector responses against F. gigantica in sheep and that the Indonesian thin tail sheep, by downregulating IgG2 responses, have an enhanced capacity for killing F. gigantica in vivo.

Cefuroxime resistance in Klebsiella pneumoniae. Susceptibility to cefotaxime and ceftazidime despite production of ESBLs.

The production of beta-lactamases, the outer membrane protein (OMP) patterns, some clinical impacts and the prevalence of resistance among cefuroxime-resistant Danish clinical isolates of Klebsiella pneumoniae were investigated. Fifteen resistant and five susceptible strains were collected from 14 patients during 1991-1994. Isolates from five patients produced extended-spectrum beta-lactamases (ESBLs). Cefuroxime resistance was accompanied by a 10-fold elevation of ciprofloxacin minimal inhibitory concentration (MIC), and for some isolates by an alteration of the OMP pattern. The relationship between alterations of the OMP patterns and cross-resistance to ciprofloxacin and the other antibiotics tested was not universal. Ten of the cefuroxime-resistant strains had elevated MICs of cefotaxime or ceftazidime, but the MICs were still below the breakpoint for susceptibility. The MICs of imipenem were not affected. Nosocomial infection or long-term colonization with resistant strains may be of importance since five patients were not treated with cefuroxime prior to isolation of the resistant strain, and all patients had either serious diseases or stayed at the hospital for a long period of time. The prevalence of cefuroxime and ciprofloxacin resistance among clinical isolates from Copenhagen county during 1990-1995 was 8.3% and 7.5%, respectively, but higher for urinary tract specimens. A greater consumption of cefuroxime as compared to cefotaxime and ceftazidime in this study, as seen generally in Denmark, indicated that ESBLs produced by the investigated strains of K. pneumoniae may be selected with cefuroxime.

Twenty-three years of Klebsiella phage typing: a review of phage typing of 12 clusters of nosocomial infections, and a comparison of phage typing with K serotyping.

OBJECTIVE: To review phage typing of 12 clusters of nosocomial Klebsiella infections which occurred between 1974 and 1997, and to compare phage typing and K serotyping. Materials and methods A total of 489 clinical and laboratory Klebsiella isolates were phage typed using 110 different phage preparations and K typed by counter current immunoelectrophoresis against 77 K antisera. RESULTS: A total of 152 phage types (PT) and 82 K types were found. Thirty-six phage types and 14 K types were represented only by the reference type strains. Of the remaining 68 K types, 60 could be subdivided into from two to 10 phage types. Ten out of 12 clusters of nosocomial Klebsiella infections could be verified as outbreaks by phage typing, whereas two clusters were found to be accumulations of sporadic cases. K typing performed retrospectively confirmed these results. In addition, for a subset of 104 epidemiologically unrelated isolates, O typing and pulsed field gel electrophoresis typing data were available. Based on these results the discriminative power of phage typing was found to be comparable with that of K typing, but phage types were less stable and reproducible. CONDITIONS: In an outbreak situation, phage typing was found to be very useful, although it seems less suitable for long-term surveillance purposes.

Direct identification and susceptibility testing of enteric bacilli from positive blood cultures using VITEK (GNI+/GNS-GA).

OBJECTIVE: To study the possibility of reporting results of identification and susceptibility testing of Gram-negative bacilli the same day as bacteremia is detected by using direct inoculation from positive blood cultures (Bactec 9240) into VITEK GNI+ and GNS-GA cards. METHODS: All blood cultures with Gram-negative enteric bacillus-like morphology on microscopy found to be positive on workdays between 15 June 1999 and 29 February 2000 were included. Identification and susceptibility testing were done by three methods: the direct method using a suspension made by differential centrifugation of positive blood culture broth for inoculation of the VITEK cards; the standard method using an inoculum made from an overnight culture on a solid media; and the routine method (reference method) using conventional testing. RESULTS: Of 169 isolates, the direct method resulted in 75% correct identifications, 9% misidentifications and 17% non-identifications. All misidentified isolates were Escherichia coli, of which 80% were reported as Salmonella arizonae. Five biochemical tests yielded most of the aberrant results; correcting the citrate and malonate reactions in most cases led to correct identification by the VITEK database. Despite a negative H2S reaction, 11 E. coli isolates were reported as S. arizonae. Two-thirds (69%) of identifications were reported within 6 h, and 95% of these were correct. The direct susceptibility testing method was assessable for 140 isolates. Correct results were found in 99% of isolate-antimicrobial combinations, and 85% were reported within 6 h. CONCLUSION: The direct VITEK method could correctly report identifications and susceptibility patterns within 6 h, making same-day reporting possible for almost two-thirds (63%) of bacteremic episodes with Gram-negative bacilli. These results could probably be improved by modification of the identification algorithms of the VITEK software.

Klebsiella typing: pulsed-field gel electrophoresis (PFGE) in comparison with O:K-serotyping.

OBJECTIVE: To compare pulsed-field gel electrophoresis (PFGE) typing and O:K-serotyping of Klebsiella in two different epidemiological settings. METHODS: One hundred and four bacteremia isolates without known epidemiological relation and 47 isolates from an outbreak in a neonatal intensive care unit (NICU) were K-typed by countercurrent immunoelectrophoresis (CCIE), O-typed by an inhibition enzyme-linked immunosorbent assay method, and typed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. RESULTS: Typing data for the 104 bacteremia isolates were compared with regard to typability, number of types, maximum number of isolates per type, and the Discriminative Index (DI). O-typing combined with K-typing (DI 0.98) as O:K-serotyping (DI 0.99) gave a very discriminative typing system, whereas O-typing alone was not very discriminative (DI 0.76). PFGE (DI 1.00) was a more discriminative typing method than O:K-serotyping, as it could subdivide 13/22 O:K-serotypes into smaller groups. Isolates with the same PFGE-type had the same O:K-serotype, indicating that isolates with different O- and/or K-types could be expected to be of different PFGE-types. Typing of the 47 isolates from the outbreak in the NICU showed that 38 isolates belonged to a single clone, and that during an epidemic limited in time and space, differences in the electrophoretic patterns of up to five bands between a parental pattern type and a subtype may be found in the PFGE profiles. CONCLUSIONS: Both O:K-serotyping and PFGE typing are highly discriminative typing methods. PFGE is the most discriminative method and is excellent for typing outbreaks with few isolates. If large numbers of isolates are to be typed, a more convenient strategy might be first to K- or O:K-serotype isolates followed by PFGE typing of possible identical isolates. Since K- or O:K-serotyping is a definitive typing method, while PFGE typing is a comparative one, PFGE cannot, for the time being, replace O:K-serotyping for surveillance purposes.

Epidemiology of Klebsiella bacteraemia: a case control study using Escherichia coli bacteraemia as control.

Epidemiological data from 117 episodes of Klebsiella bacteraemia were compared with those from matched controls with Escherichia coli bacteraemia. Cases and controls were obtained from 20,631 blood cultures taken from patients in Hvidovre Hospital between 1990 and 1992. The data studied included: sex and age, risk factors, portal of entry, outcome, nosocomial acquisition and distribution within the hospital. The incidence of Klebsiella bacteraemia was 9.3/10,000 admissions (76% Klebsiella pneumoniae; 24% Klebsiella oxytoca). Patients with Klebsiella and E. coli bacteraemia had many common features, including a high incidence of neoplastic disease, biliary tract disease, and renal failure. Many had undergone surgery or received therapy with steroids, antacids or antibiotics. Klebsiella bacteraemia was more often found in males, in patients with hospital contact within the previous month, and polymicrobial infection. Logistic regression analysis showed that use of invasive plastic devices and diabetes were significantly associated with Klebsiella bacteraemia. The urinary tract was the commonest source, followed by the biliary tract; 27% of patients had no obvious focus of infection, and in many of these an invasive device may have been involved. Forty-five K-serotypes were found--the largest number being nine strains of type K3; only a few strains had acquired resistance characters to antimicrobial agents. There were no differences between community- and hospital-acquired strains; indicating that our hospital does not have a resident strain of Klebsiella.

[Familial occurrence of Chlamydia pneumoniae infection]. / Familiaer forekomst af Chlamydia pneumoniae-infektion.

The clinical courses of six patients involved in a family outbreak of Chlamydia pneumoniae respiratory tract infection are described. The diagnosis was established by use of culture, polymerase chain reaction and determination of species specific antibodies. The patients had mild influenza-like symptoms with sore throat, occluded eustachian tubes and long-lasting cough. All patients received recommended antibiotic treatment regimens. Two out of the six patients needed further antibiotic treatment to obtain clinical and microbiological cure.

[Extended spectrum beta-lactamases in Danish Klebsiella isolates]. / Betalaktamaser med udvidet spektrum hos danske Klebsiella-isolater.

This study presents the first two cases of infections with Klebsiella pneumoniae producing extended spectrum betalactamases (ESBL) that have been recorded in Denmark. They presented as a urinary tract infection and a generalized infection in a patient admitted to an intensive care unit. Both patients had been treated with broad spectrum antibiotics prior to infection. Presumably, one of the strains had been imported from Turkey. The ESBL of the two strains were characterized as SHV-2 and SHV-5, respectively. Patients transferred from hospitals abroad should be screened for Klebsiella producing ESBL, in addition to MRSA and other multiresistant organisms. A restrictive antibiotic policy and strict hygienic precautions are essential measures to control the selection and spread of such organisms in the hospital environment.

The flagellar fraction of Trypanosoma cruzi depleted of an immunosuppressive antigen enhances protection to infection and elicits spontaneous T cell responses.

The flagellar fraction (FF) of Trypanosoma cruzi can be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF-beta (Hansen et al., submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom-matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice. In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL-1 and IL-6. In immunized mice, the FT elicited higher levels of antigen-specific IgG2a than the FF as well as broader recognition of T. cruzi antigens. Splenocytes from mice immunized with FT proliferated spontaneously in vitro and secreted TH1 and TH2 cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN-gamma. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN-gamma through a TGF-beta dependent mechanism.

An immunoaffinity-purified Trypanosoma cruzi antigen suppresses cellular proliferation through a TGF-beta-mediated mechanism.

Two subfractions with opposite immunological properties were obtained from the flagellar antigens (FF) of Trypanosoma cruzi epimastigotes by immunoaffinity chromatography. The ligand-bound material (Ag 123) contained four polypeptide bands of 97, 55, 38 and 14 kDa. The nonretained flow-through (FT), induced a potent proliferation of murine naive splenocytes. In contrast, Ag 123 inhibited the proliferative capacity of the FT as well as the proliferation mediated by the mitogen Concanavalin A (Con A). The suppressive effect of Ag 123 on the Con A-mediated proliferation was neutralized by an anti-TGF-beta monoclonal antibody. Both Ag 123 and FF stimulated high serum levels of TGF-beta in injected mice. Ag 123 also induced in vitro secretion of TGF-beta by murine splenocytes. These results demonstrate that Ag 123 is a potent stimulator of TGF-beta both in vivo and in vitro. Oligopeptides derived from the 38 kDa protein present in Ag 123 showed homology with human and rat alpha-fetoproteins (AFP). Ag 123 seems to have a key role in the immunosuppression that develops during early stages in the infection with T. cruzi.

Serospecificity of a cloned protease-resistant Treponema pallidum--specific antigen expressed in Escherichia coli.

We evaluated the serological reactivity of a protease-resistant antigen designated 4D which was encoded by Treponema pallidum DNA and was expressed in Escherichia coli from recombinant plasmid pAW329. This 19,000-molecular-weight antigen was purified in its native, non-protease-treated form from E. coli sonic extracts by molecular sieving and ion-exchange chromatography. Antibody binding to antigen 4D was detected by a radioimmunoassay. Antigen 4D-specific antibody was detected in 95% of the sera in a Centers for Disease Control syphilis serum panel. It was also detected in 55% of 121 primary syphilis patients, whereas syphilis antibody was detected in 83% of the sera by a fluorescent treponemal antibody absorption test and in 88% of the sera by a T. pallidum microhemagglutination test. In tests of 118 normal sera, less than 3% demonstrated antibody to antigen 4D; these results are similar to microhemagglutination and fluorescent treponemal antibody absorption test results. Rabbit antisera against Treponema phagedenis, Treponema refringens, Treponema denticola, and Treponema vincentii did not react with antigen 4D.

Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniae clinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+ and half O- strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.

Somatic serogroups, capsular types, and species of fecal Klebsiella in patients with ankylosing spondylitis.

The purpose of the present study was to find out whether patients with ankylosing spondylitis (AS) carry fecal Klebsiella strains that belong to serotypes or species specific for AS. Somatic serotypes (O groups), capsular (K) serotypes, and biochemically identified species were determined for fecal klebsiellae isolated from 187 AS patients and 195 control patients. The controls were patients with fibromyalgia or rheumatoid arthritis. The 638 isolates of Klebsiella that were obtained represented 161 strains; 81 from AS patients and 80 from the controls. The average number of Klebsiella strains per patient was 1.7 for the AS group and 1.5 for the control group. The most common O group was O1, which was observed for isolates from 23 of 187 AS patients and 24 of 195 control patients. Next in frequency was group O2, which was observed for isolates from 17 AS patients and 15 control patients. Regarding the K serotypes, 59 different types were identified, revealing a heterogeneous representation of Klebsiella strains, without a predominance of any serotype. By biochemical identification, Klebsiella pneumoniae was the most frequently occurring species, being found in 45 AS patients and 45 control patients. Next in the frequency was K. oxytoca, which was observed in 26 AS patients and in 29 control patients. K. planticola and K. terrigena occurred in only a minority of patients. Altogether, when analyzed either separately or simultaneously according to O groups, K serotypes, and biochemically identified species, no evidence of the existence of AS-specific Klebsiella strains was obtained. These findings do not indicate participation of Klebsiella in the etiopathogenesis of AS.

A case of Moraxella canis-associated wound infection.

Moraxella canis was isolated from an infected foot ulcer in a patient suffering from diabetes mellitus with neuropathy. Bacteriological findings and 16S rDNA data are presented.

Impairment of respiratory burst in polymorphonuclear leukocytes by extended-spectrum beta-lactamase-producing strains of Klebsiella pneumoniae.

The ability of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing Klebsiella pneumoniae strains to induce a respiratory burst in polymorphonuclear leukocytes (PMNLs) was investigated. Ninety ESBL-producing and 178 non-ESBL-producing Klebsiella pneumoniae isolates were serotyped and their ability to induce a respiratory burst in PMNLs tested by monitoring the cells' chemiluminescence (CL) response. The percentage of isolates inducing high levels of CL response (CL>75%) was significantly higher among non-ESBL producers (52%) than among ESBL producers (32.2%) ( P<0.0001; OR=3.396; 95%CI=2.036-5.664). The median CL response was significantly higher among the non-ESBL producers (76.9%) than among the ESBL producers (52.6%) ( P=0.034). The two groups did not differ in their ability to resist intracellular killing by PMNLs ( P>0.05), with strains inducing high levels of CL response having significantly lower survival rates (31.8% vs. 42.4%) than strains inducing low levels of CL response (164% vs. 200%) ( P<0.01). The frequencies of the K2 and the K25 serotypes were significantly higher among ESBL-producing strains (17.8% and 22.2%, respectively) than among the non-ESBL producers (6.2% and 1.7%, respectively) ( P=0.0057 and P<0.0001). Of the 77 Klebsiella K serotypes, 71 were detectable among the non-ESBL producers, but only 24 were detectable among the ESBL producers. ESBL-producing Klebsiella pneumoniae strains might have a greater pathogenic potential by virtue of their ability to escape the phagocytic activity of PMNLs.

Increased serum resistance in Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases.

The aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae. Ninety ESBL-producing and 178 non-ESBL-producing K. pneumoniae isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (P = 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both Klebsiella groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (P = 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (P = 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.