Anne von Gottberg

Rapid Pneumococcal Evolution in Response to Clinical Interventions

Streptococcus pneumonia evades vaccines and drugs by high levels of recombination and rapid adaptation. Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain23F-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

Outcome of Cephalosporin Treatment for Serious Infections Due to Apparently Susceptible Organisms Producing Extended-Spectrum β-Lactamases: Implications for the Clinical Microbiology Laboratory

ABSTRACT Although extended-spectrum beta-lactamases (ESBLs) hydrolyze cephalosporin antibiotics, some ESBL-producing organisms are not resistant to all cephalosporins when tested in vitro. Some authors have suggested that screening klebsiellae or Escherichia colifor ESBL production is not clinically necessary, and when most recently surveyed the majority of American clinical microbiology laboratories did not make efforts to detect ESBLs. We performed a prospective, multinational study of Klebsiella pneumoniae bacteremia and identified 10 patients who were treated for ESBL-producingK. pneumoniae bacteremia with cephalosporins and whose infecting organisms were not resistant in vitro to the utilized cephalosporin. In addition, we reviewed 26 similar cases of severe infections which had previously been reported. Of these 36 patients, 4 had to be excluded from analysis. Of the remaining 32 patients, 100% (4 of 4) patients experienced clinical failure when MICs of the cephalosporin used for treatment were in the intermediate range and 54% (15 of 28) experienced failure when MICs of the cephalosporin used for treatment were in the susceptible range. Thus, it is clinically important to detect ESBL production by klebsiellae or E. coli even when cephalosporin MICs are in the susceptible range (≤ 8 μg/ml) and to report ESBL-producing organisms as resistant to aztreonam and all cephalosporins (with the exception of cephamycins).

International Prospective Study of Klebsiella pneumoniae Bacteremia: Implications of Extended-Spectrum β-Lactamase Production in Nosocomial Infections

Context Worldwide prevalence of extended-spectrum -lactamase (ESBL)producing organisms is of great concern because of their broad antibiotic resistance. Contribution Analysis of consecutive cases of Klebsiella pneumoniae bacteremia at 12 hospitals on 6 continents shows that although incidence varies widely among institutions, almost one third of cases of nosocomial bacteremia and almost one half of intensive care unitbased infections were caused by ESBL-producing organisms. Patient-to-patient spread is common, and prevention requires careful attention to routine infection control measures. Cautions Specific antibiotic exposures before K. pneumoniae infection cannot be confirmed as risk factors for ESBL-related infections on the basis of this study. The Editors Since the discovery that resistance of Staphylococcus aureus to penicillin is mediated by a -lactamase, much effort has been made to create -lactam antibiotics that are stable to common -lactamases. Cephalosporin antibiotics containing an oxyimino side-chain represent a major advance in antibiotic development. The merger of the oxyimino chain and a 2-amino-5-thiazolyl nucleus (in such antibiotics as ceftriaxone, cefotaxime, and ceftazidime) resulted in stability to the effects of the common TEM-1 and SHV-1 -lactamases produced by gram-negative bacilli, such as Escherichia coli and Klebsiella pneumoniae. However, within a few years of the commercial release of these antibiotics, gram-negative bacilli (especially K. pneumoniae) that harbored mutated versions of the parent TEM and SHV enzymes were detected. These and other newly detected -lactamases (for example, the functionally similar CTX-M types) hydrolyze -lactam antibiotics containing the oxyimino side-chain. Genes encoding these extended-spectrum -lactamases (ESBLs) were carried on transferable plasmids. These plasmids frequently carried determinants of resistance to other classes of antibiotics, particularly the aminoglycosides (1, 2). Thus, ESBL-producing gram-negative bacilli were found to truly be multiresistant pathogens: The majority of these strains were resistant to all -lactam antibiotics (with the exception of cephamycins and carbapenems), most aminoglycosides, trimethoprimsulfamethoxazole, and sometimes the fluoroquinolones. Although the prevalence of ESBL production among gram-negative bacilli varies geographically (and may even vary from hospital to hospital within a city), widespread recognition of the advent of these -lactamases has been lacking (3). Previous studies of the epidemiology of ESBL-producing organisms have been largely limited to single institutions (4, 5). Because substantial information on the epidemiology of ESBL-producing K. pneumoniae or other gram-negative bacilli is lacking, we established a collaboration of researchers from 7 countries on 6 continents to prospectively enroll consecutive patients with K. pneumoniae bacteremia. Methods Study Design We performed a prospective observational study of 440 consecutive, sequentially encountered patients with K. pneumoniae bacteremia at 12 hospitals in South Africa, Taiwan, Australia, Argentina, the United States, Belgium, and Turkey. No patient was excluded from analysis. Patients were enrolled from 1 January 1996 to 31 December 1997. Patients were older than 16 years of age and had positive blood cultures for K. pneumoniae. The investigators completed a 188-item study form on each episode of K. pneumoniae bacteremia. Patients were followed for 1 month after the onset of bacteremia to assess clinical outcome, including death and infectious complications. The study was observational in that administration of antimicrobial agents and other therapeutic management was controlled by the patients physician rather than the investigators (6). The study was approved by institutional review boards as required by local hospital policy at the time of the study. Definitions All study definitions were established before data analysis. Nosocomial bacteremia was defined as K. pneumoniae bacteremia occurring more than 48 hours after admission to hospital. An episode of bacteremia was defined as the period of 14 days from the time of collection of the first blood culture positive for K. pneumoniae. Severity of acute illness was assessed at the time of the positive blood cultures by using the Pitt bacteremia score, a previously validated scoring system that is based on mental status, vital signs, requirement for mechanical ventilation, and recent cardiac arrest (Table 1) (7, 8). Severity of illness in patients in an intensive care unit at the time of onset of bacteremia was assessed by using the Acute Physiology and Chronic Health Evaluation-3 score (9). Site of infection was determined to be pneumonia, urinary tract infection, meningitis, incisional wound infection, other soft-tissue infection, intra-abdominal infection, or primary bloodstream infection according to Centers for Disease Control and Prevention definitions (10). Previous antibiotic therapy was defined as antibiotics given for at least 2 days within the 14 days before an episode of K. pneumoniae bacteremia (11). Antibiotic therapy for the episode of K. pneumoniae bacteremia was the receipt of antibiotics that are active in vitro against the blood culture isolate (that is, susceptible according to 1999 NCCLS breakpoints [12], given for at least 2 days within the first 5 days of collection of the first positive blood culture. Mortality was death from any cause within 14 days from the date of the first positive blood culture for K. pneumoniae. Table 1. The Pitt Bacteremia Score* Microbiological Analysis Production of ESBL was phenotypically determined by broth dilution using the NCCLS performance standards current as of January 1999 (12). A 3 twofold decrease in the minimal inhibitory concentration (MIC) for cefotaxime or ceftazidime tested in combination with clavulanic acid compared with its MIC when tested alone was considered phenotypic confirmation of ESBL production. For example, an isolate with a ceftazidime MIC of 8 g/mL and a ceftazidimeclavulanic acid MIC of 1 g/mL fulfills this definition of an ESBL-producing organism (12). The MICs of antibiotics commonly used in the treatment of gram-negative sepsis were determined for the ESBL-producing isolates by using the gradient diffusion method (Etest, AB Biodisk, Solna, Sweden). Pulsed-field gel electrophoresis was used to establish the genotypic relationships of ESBL-producing isolates from each hospital (11). Statistical Analysis All statistical comparisons were made by using PROPHET Statistics software, version 6.0 (ABTech-BBN Corp., Charlottesville, Virginia) or Stata software, version 7.0 (Stata Corp., College Station, Texas). For bivariate comparisons, the chi-square or Fisher exact test was used to compare categorical variables. Continuous variables were compared by using the t-test or the MannWhitney test. Length of stay before positive blood culture was missing for 10 (4%) patients, all of whom had been readmitted within 1 week of discharge. Length of stay for these 10 patients was therefore estimated by using a predictive model that assumed missing at random. Because previous receipt of antibiotics was not random, a propensity score model was used to further evaluate the effect of receipt of antibiotics containing an oxyimino group as a risk for ESBL production. The score was calculated by using a logistic model in which receipt of -lactam antibiotics containing an oxyimino group (cefuroxime, ceftriaxone, cefotaxime, ceftazidime, or aztreonam) was the dependent variable (scored as yes or no) and predictors were all available factors hypothesized to influence the receipt of antibiotic therapy (underlying patient conditions), with adjustment for center by using the indicator variables for site. Factors included in the calculation of this score were age, sex, admission from nursing home, underlying diseases (cancer, HIV infection, diabetes, or renal and liver disease), previous surgery, use of corticosteroids, presence of a central line, mechanical ventilatory support, presence of a nasogastric tube, and the indicator variables for site. A logistic model clustered on the patient that used receipt of antibiotics containing an oxyimino group and the quintile stratified score was then used to evaluate the risk for ESBL production. Role of the Funding Source Merck and Company provided support for laboratory studies but played no role in study design, conduct of the study, interpretation of the results, or approval of the study before publication. Results During the study period, 455 episodes of K. pneumoniae bacteremia occurred in 440 patients; of these, 202 (44.4%) episodes in 196 patients were community acquired and 253 (55.6%) episodes in 244 patients were nosocomially acquired. Table 2 shows the characteristics of the participants. Table 2. Characteristics of 244 Patients with Nosocomial Klebsiella pneumoniae Bacteremia Of the episodes of K. pneumoniae bacteremia, 18.7% (85 of 455) were due to ESBL-producing organisms and 81.1% (369 of 455) were due to nonESBL-producing organisms. One additional episode involved an isolate that showed a markedly elevated MIC for the oxyimino -lactam antibiotics but no decrease in MIC with the addition of clavulanic acid. This isolate had an MIC for cephamycin antibiotics in the resistant range and may have possessed an AmpC-like enzyme. This episode was excluded from further analysis. Seventy-eight of 253 (30.8%) episodes of nosocomial bacteremia were due to ESBL-producing organisms. Table 3 shows the sites of infection associated with nosocomial ESBL-producing K. pneumoniae bacteremia. Fewer episodes of community-acquired K. pneumoniae bacteremia (3.5% [7 of 202]) were due to ESBL-producing organisms (P <0.001) (risk ratio, 8.9 [95% CI, 4.2 to 18.8]). Of the 253 episodes of nosocomial bacteremia, 69 were acquired in the intensive care unit. Of these 69 episodes, 30 (43.5%) episodes o

Antibiotic Therapy for Klebsiella pneumoniae Bacteremia: Implications of Production of Extended-Spectrum β-Lactamases

The prevalence of extended-spectrum beta -lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of 455 consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL-producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem (primarily imipenem) was associated with a significantly lower 14-day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

Epidemiology of Ciprofloxacin Resistance and Its Relationship to Extended-Spectrum β-Lactamase Production in Klebsiella pneumoniae Isolates Causing Bacteremia

A prospective study of Klebsiella pneumoniae bacteremia was performed in 12 hospitals in 7 countries. Of 452 episodes of bacteremia, 25 (5.5%) were caused by K. pneumoniae that was resistant in vitro to ciprofloxacin. Extended-spectrum beta-lactamase (ESBL) production was detected in 15 (60%) of 25 ciprofloxacin-resistant isolates, compared with 68 (16%) of 427 ciprofloxacin-susceptible strains (P=.0001). Multivariate analysis revealed that risk factors for ciprofloxacin resistance in K. pneumoniae included prior receipt of a quinolone (P=.0065) and an ESBL-producing strain (P=.012). In all, 18% of ESBL-producing isolates were also ciprofloxacin-resistant. Pulsed-field gel electrophoresis showed that 11 of the 15 ciprofloxacin-resistant ESBL-producing strains belonged to just 4 genotypes, suggesting that patient-to-patient transmission of such strains occurred. The close relationship between ESBL production and ciprofloxacin resistance is particularly worrisome because the first reported instance of plasmid-mediated ciprofloxacin resistance has been in an isolate of K. pneumoniae also possessing an ESBL.

Community-Acquired Klebsiella pneumoniae Bacteremia: Global Differences in Clinical Patterns

We initiated a worldwide collaborative study, including 455 episodes of bacteremia, to elucidate the clinical patterns of Klebsiella pneumoniae. Historically, community-acquired pneumonia has been consistently associated with K. pneumoniae. Only four cases of community-acquired bacteremic K. pneumoniae pneumonia were seen in the 2-year study period in the United States, Argentina, Europe, or Australia; none were in alcoholics. In contrast, 53 cases of bacteremic K. pneumoniae pneumonia were observed in South Africa and Taiwan, where an association with alcoholism persisted (p=0.007). Twenty-five cases of a distinctive syndrome consisting of K. pneumoniae bacteremia in conjunction with community-acquired liver abscess, meningitis, or endophthalmitis were observed. A distinctive form of K. pneumoniae infection, often causing liver abscess, was identified, almost exclusively in Taiwan.

Virulence Characteristics of Klebsiella and Clinical Manifestations of K. pneumoniae Bloodstream Infections

Differences in clinical manifestations are due to virulence factors expressed by the organism.

Sequence Diversity of the Factor H Binding Protein Vaccine Candidate in Epidemiologically Relevant Strains of Serogroup B Neisseria meningitidis

BACKGROUND Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

Pneumococcal Capsular Switching: A Historical Perspective

Background.Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. Methods.Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events. Results.We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a. Conclusions.Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.

Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens

ABSTRACT Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.