The prevalence and characteristics of Streptococcus pneumoniae isolates expressing serotypes 6C and 6D in Hong Kong prior to the introduction of the 7-valent pneumococcal conjugate vaccine

A study was conducted to determine the prevalence of the 2 newly described types, 6C and 6D, among pneumococcal isolates collected in Hong Kong before availability of the 7-valent pneumococcal conjugate vaccine. A total of 154 serogroup 6 isolates obtained from nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2) during 1995 to 2001 were analyzed by polymerase chain reaction typing. Five nasopharyngeal and 2 sputum isolates were found to belong to 6C and 6D, respectively. The isolates were genetically diverse, but one 6C and two 6D isolates exhibited some clonal relationship. Phylogenetic analysis of the wchA-wciN(β)-wciO nucleotide sequences showed that the Hong Kong 6C/6D isolates had 2 allelic profiles, which were more closely related to 6C/6D isolates from Fijian and Korea than were those from Brazil and the United States. However, all of the wciP gene sequences for both Hong Kong and non-Hong Kong isolates clustered together: 6C isolates with the wciP-9 allele and 6D isolates with the wciP-5 allele. In conclusion, the prevalence of the 2 newly described serotypes was low before the era of the pneumococcal conjugate vaccine. Nonetheless, results from the molecular studies indicated that the evolution of the capsular genes have involved complex pathways.     

Prevalence of penicillin-nonsusceptible pneumococcal bacteremia in a Staten Island community hospital

BACKGROUND: Although the first reports of infection due to penicillin-nonsusceptible Streptococcus pneumoniae in the United States were in children, these strains have circulated widely in recent years, with the prevalence increasing dramatically among the elderly. Regional surveillance of pneumococcal susceptibility profiles may assist clinicians in management decisions, increase awareness of this microbial threat, and target potential areas of intervention. METHODS: As part of ongoing surveillance, we surveyed single-patient pneumococcal blood isolates in our 440-bed Staten Island community teaching hospital from June 1, 1996, through May 31, 1998. RESULTS: Overall, of 47 single-patient isolates, 16 (35%) were penicillin nonsusceptible. Of 35 isolates from adults, 15 (44%) were nonsusceptible, compared with 1 of 12 (8%) from children. Seven of the nonsusceptible isolates (44%) were from persons > or = 65 years old and represented 47% of the isolates from this age group. CONCLUSIONS: Community-acquired penicillin-nonsusceptible pneumococcal bacteremia is not simply a pediatric problem, but also a threat to the elderly.     

Comparison of methods for detection of Streptococcus pneumoniae serotype 6C

Polymerase chain reaction-based methods and Quellung method were compared for serotyping of Streptococcus pneumoniae serogroup 6 for 230 isolates from Asian countries. Prevalence of serotype 6C differed according to the method used, with inconsistencies in 12 pneumococcal isolates (5.2%). Thus, evaluation of the prevalence of serotype 6C may differ according to the methods used.     

Use of 2 pneumococcal common protein real-time polymerase chain reaction assays in healthy children colonized with Streptococcus pneumoniae

Polymerase chain reaction (PCR) offers promise in pneumococcal diagnostics, but whether nasopharyngeal carriage causes false-positive results in people colonized with Streptococcus pneumoniae is unknown. We found in serum no positive samples for pneumococcal DNA in 100 carriers and noncarriers using 2 different real-time PCR assays targeting lytA and psaA genes.     

Prevalence, mechanisms, and risk factors of carbapenem resistance in bloodstream isolates of Pseudomonas aeruginosa

We examined the prevalence of various carbapenem resistance mechanisms in Pseudomonas aeruginosa bloodstream isolates from a university-affiliated hospital. Isolates obtained in 2003 and 2004 were screened for meropenem/imipenem resistance, and clonality was assessed by repetitive-element-based polymerase chain reaction. The presence of carbapenemase and AmpC overexpression was ascertained by spectrophotometric assays. Outer membrane protein profiles were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and efflux pump overexpression was confirmed by Western blotting. We examined 129 nonrepeat isolates; 21 isolates (from 13 distinct clones) were resistant to meropenem or imipenem (prevalence rate = 16.3%). Nineteen (90.5%) carbapenem-resistant isolates had reduced OprD expression, and 6 (28.6%) isolates had overexpression of MexB. Increased length of hospital stay was identified as a significant risk factor for bacteremia due to carbapenem-resistant P. aeruginosa. Understanding the prevalence and mechanism of carbapenem resistance in P. aeruginosa may guide empiric therapy for nosocomial infections in our hospital.     

Pharmacokinetics of sparfloxacin and serum bactericidal activity against pneumococci.

Sparfloxacin, a new fluorinated quinolone, exhibits higher in vitro activity against pneumococci than do ciprofloxacin and ofloxacin. Since up to 30% of cases of pneumococcal pneumonia are associated with bacteremia, and since an increasing percentage of pneumococci are resistant against penicillin, we studied the serum bactericidal activity of sparfloxacin against pneumococci in eight healthy, middle-aged volunteers. Pharmacokinetics in serum and urine after a 400-mg oral dose of sparfloxacin were comparable to those described by other authors. Inhibitory and bactericidal activities in serum were measured for four pneumococcal isolates representing penicillin-susceptible (one isolate), intermediately resistant (two isolates), and highly resistant (one isolate) strains. Geometric mean inhibitory titers ranged between 1:2.4 and 1:6.3 and bactericidal titers ranged between 1:1.3 and 1:3.6 during a time period of 1 to 6 h after drug intake. Although such titers were not sufficient to predict a clinical response based on previous pharmacodynamic studies using quinolone antibiotics, data obtained with volunteers may only partially reflect the clinical situation in which a rise of humoral antibodies directed against pneumococcal antigens may help to reinforce the bactericidal action of the antibiotic.     

Changes in the epidemiology of pneumococcal bacteremia in a Swiss university hospital during a 15-year period, 1986-2000

OBJECTIVE: To evaluate changes in epidemiological characteristics and outcome of patients with pneumococcal bacteremia during a 15-year period in a Swiss university hospital. PATIENTS AND METHODS: We reviewed the medical records of all hospitalized adults at the University Hospital Basel, Basel, Switzerland, whose blood culture yielded Streptococcus pneumoniae from January 1, 1986, through December 31, 2000. RESULTS: We analyzed 405 episodes of pneumococcal bacteremia in 394 patients. The mean annual incidence of 1.78 episodes per 1000 hospital admissions was inversely related to the mean atmospheric temperature of the area. During the study period, penicillin nonsusceptibility increased from 0% to 17%. The overall case-fatality rate was 25%, which decreased from 33% to 17% between the first and the second half of the study period (P<.001). The proportion of women with pneumococcal bacteremia increased from 37% to 52%. Independent risk factors for fatal outcome were coronary artery disease (P<.001; relative risk [RR], 4.3; 95% confidence interval [CI], 3.4-5.1), neutropenia (P=.001; RR, 3.2; 95% CI, 1.9-4.8), and age 65 years or older (P=.001; RR, 2.9; 95% CI, 1.8-4.2), whereas prior respiratory tract infection (P=.03; RR, 0.3; 95% CI, 0.1-0.5) and the occurrence of pneumococcal bacteremia in the second half of the study period (P=.01; RR, 0.4; 95% CI, 0.2-0.6) were independent predictors of survival. The case-fatality rate in human immunodeficiency virus (HIV)-infected patients was significantly lower than in patients not infected with HIV or in those with unknown HIV status (9% vs 27%; P=.006), which correlated with the younger mean +/- SD age of HIV-infected patients (33.2+/-6.6 years) compared with patients not infected with HIV (63.1+/-18.1 years) (P<.001). CONCLUSIONS: The case-fatality rate of patients with pneumococcal bacteremia decreased significantly between the first and second half of the study period, despite the increased prevalence of penicillin-nonsusceptible isolates. Independent risk factors for fatal outcome were coronary artery disease, neutropenia, and age 65 years or older, whereas prior respiratory tract infection and the occurrence of pneumococcal bacteremia in the second half of the study period were independent predictors of survival. HIV infection was a predisposing factor for pneumococcal bacteremia but was not a risk factor for fatal outcome.     

Serotyping of 1,458 pneumococcal isolates with analysis of bacteremia in 84 patients

During a 61-month period, 1,458 pneumococcal isolates (including 87 bacteremia strains) were collected at the Albany Veterans Administration Medical Center and serotyped with the use of the typing system of the New York Department of Health Laboratories. Fifty percent of all isolates were of types in the 14-valent vaccine, while 68% were in 23-valent vaccine. Types 3, 6, 9, and 34 were the most prevalent types. Sixty-two percent of blood isolates were of types in the 14-valent vaccine, while 77% were in the 23-valent vaccine. Fifty-six percent of bacteremias were nosocomial and 44% were community acquired with a fatality rate of 41% and 18%, respectively. The fatality rate was greatest with risk factors such as asplenia and azotemia. These data support the recommendation for the use of 23-valent pneumococcal polysaccharide vaccine in high-risk patients, especially those who might acquire bacteremia in a nosocomial setting.     

Pneumococcal bacteremia as a marker for human immunodeficiency virus infection in patients without AIDS

We retrospectively reviewed medical records of all adult patients with blood cultures positive for Streptococcus pneumoniae to determine the number of HIV seropositive patients in whom S pneumoniae bacteremia was the presenting manifestation. We also compared the clinical presentation, laboratory data, and outcome of pneumococcal bacteremia in patients who were HIV seropositive with patients with no risk factors for HIV infection. All adult patients with blood cultures positive for S pneumoniae from January 1987 through April 1989 at two acute care general hospitals in northern California were identified by review of microbiology data. One hospital served veterans, the other the indigent of a suburban area. Six (15%) of 41 patients with pneumococcal bacteremia were HIV seropositive; five were not known to be HIV seropositive before the onset of bacteremia, and the sixth was asymptomatic with respect to HIV infection. No patient with AIDS had pneumococcal bacteremia. HIV seropositive patients were significantly younger, had significantly fewer underlying diseases, and had fewer complications of pneumococcal bacteremia than bacteremic patients with no risk factors for HIV infection. Patients with pneumococcal bacteremia should be evaluated for HIV infection, especially in the absence of other underlying diseases that predispose to pneumococcal bacteremia.     

Enterobacterial repetitive intergenic consensus PCR is a useful tool for typing Mycobacterium chelonae and Mycobacterium abscessus isolates

Outbreaks of rapidly growing mycobacterium (RGM) infections are increasingly being reported worldwide. Information about genetic relatedness of isolates obtained during outbreaks can provide opportunities for prompt intervention. Pulsed-field gel electrophoresis (PFGE) is expensive, time consuming, and labor intensive. Other than that, Mycobacterium abscessus isolates can suffer DNA degradation during electrophoresis. Polymerase chain reaction (PCR)-based methods are cheaper, faster, and easier to perform, but discriminatory power varies depending on the primer used. In this study, we tested the competence of enterobacterial repetitive intergenic consensus (ERIC) PCR in comparison with PFGE to distinguish unrelated isolates (24 Mycobacterium chelonae and 24 M. abscessus) obtained from human and/or environmental samples and to group 56 isolates from 6 outbreaks confirmed epidemiologically, caused by M. chelonae and M. abscessus after ophthalmologic refractive surgery and mesotherapy. Enterobacterial repetitive intergenic consensus PCR presented discriminatory power, calculated using Simpson's index of diversity, of 0.989 for M. abscessus and 0.975 for M. chelonae and grouped outbreak isolates in distinct groups showing epidemiologic concordance. Pulsed-field gel electrophoresis also grouped outbreak isolates and presented discriminatory power of 0.972 and 0.993 for M. abscessus and M. chelonae, respectively. DNA from 8 (22%) of 36 M. abscessus isolates analyzed showed degradation during electrophoresis. Compared with PFGE and epidemiologic information as the gold standard, ERIC PCR is a simple, high throughput, affordable, reproducible, and discriminatory molecular typing method for inference of genetic relatedness of RGMs of the M. chelonae-abscessus group.     

Use of the housekeeping genes, gdh (zwf) and gki, in multilocus sequence typing to differentiate Streptococcus pneumoniae from Streptococcus mitis and Streptococcus oralis

Polymerase chain reaction and sequencing of the housekeeping genes, gdh (zwf) and gki, based on the primers and alleles from multilocus sequence typing can be used to delineate and support the identity of clinical isolates of Streptococcus pneumoniae and differentiate from the closely related Streptococcus mitis and Streptococcus oralis.     

Prevalence and characterization of metallo-beta-lactamases in clinical isolates of pseudomonas aeruginosa

The prevalence and the type(s) of metallo-beta-lactamases (MBLs) produced by isolates of Pseudomonas aeruginosa were investigated. During 2001, 506 nonduplicate isolates were obtained from hospitalized patients. Eighty-two strains were selected because of resistance to carbapenems and/or ceftazidime. Screening for MBL production was performed in the latter isolates by the Etest MBL strips (AB Biodisk, Solna, Sweden) and by a broth microdilution method measuring minimum inhibitory concentrations (MICs) of imipenem alone and in the presence of metal-chelating agents (EDTA and o-phenanthroline). Specific DNA probes were used to investigate the presence of genes coding for IMP- or VIM-type enzymes. Overall, four isolates of P. aeruginosa (obtained from independent patients) were found to carry a blaVIM gene. Polymerase chain reaction (PCR) experiments and DNA sequencing revealed that the VIM-2 determinant was present in three cases, whereas VIM-1 was detected in one isolate. Surveillance programs should be adopted to avoid the spread of these worrisome resistance genes.     

Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene

OBJECTIVE: Bacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia. DESIGN: The results of the T-RFLP analysis are compared with two standard biochemical identification methods. METHODS: Lactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed. RESULTS: T-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations. CONCLUSION: T-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.     

A 13-year study of antimicrobial susceptibility of common gram-negative bacteria isolated from the bloodstream in a teaching hospital

The choice of antimicrobial therapy for the treatment of bacteremia is often empirical and based on the knowledge of susceptibility profiles of the most common bacteria. We analyzed blood culture isolates from a teaching hospital for 13 years prospectively. This study examined the susceptibility profiles of 6,616 gram-negative bacteria. Escherichia coli ranked among the commonest bacteria, representing 43.6% (2,890) of all gram-negative isolates. Klebsiella sp. ranked second, 17.7% (1,171) and 16.7% were resistant to ceftazidime in 1997. There was a trend towards an increase in resistance to fluoroquinolones in the common gram-negative bacteria. Imipenem was the most active agent against gram-negative bacteria. The results of the susceptibility of gram-negative bacteria causing bacteremia provide valuable information for implementing the appropriate chemotherapy for bacteremia.     

同源重组敲除临床肺炎克雷伯菌质粒bla_(KPC-2)基因

目的建立敲除临床肺炎克雷伯菌耐药菌株质粒上的耐药基因的方法。方法聚合酶链反应(PCR)分别扩增试验菌株待敲除目的基因blaKPC-2的上下游片段及质粒pMQ300的潮霉素耐药基因片段,采用重叠延伸基因扩增(SOE-PCR)技术构建融合片段,以耐阿普霉素的λred质粒pKOBEG-Apr为介导,同源重组敲除临床肺炎克雷伯菌耐药菌株的blaKPC-2基因。用含潮霉素和阿普霉素的LB培养基筛选重组子,用PCR和逆转录PCR扩增检测blaKPC-2基因、潮霉素耐药基因及药物敏感性验证重组子。结果不同多位点序列分析(MLST)型别的2株临床肺炎克雷伯菌耐药菌株的blaKPC-2基因得以敲除。结论λred同源重组法可以可靠敲除临床肺炎克雷伯菌耐药菌株质粒上的基因。     

Retrograde transmission of Proteus mirabilis during platelet transfusion and the use of arbitrarily primed polymerase chain reaction for bacteria typing in suspected cases of transfusion transmission of infection

BACKGROUND: When bacteria are found, after a platelet transfusion, in the recipient's blood as well as in the platelet concentrate (PC), a causal relationship is normally suspected, with the PC as the causative agent. The other alternative, that the patient has bacteremia and contaminated the PC, is less well documented in the literature. CASE REPORT: Arbitrarily primed polymerase chain reaction (AP-PCR) was used for testing strains of Proteus mirabilis isolated from a patient's blood before and after a platelet transfusion and from the PC. Because of a febrile reaction after a platelet transfusion, bacterial culture was performed on the PC used, showing growth of P. mirabilis. The same species was found in the patient's blood after the transfusion. Posttransfusion sepsis caused by a contaminated PC was suspected, and anti-sepsis treatment was given to the recipient. Later, it became apparent that the patient had had bacteremia before the transfusion and that P. mirabilis was one of the species in the isolate. With AP-PCR, the identity of the three P. mirabilis isolates could be distinguished. CONCLUSION: AP-PCR is a useful technique for distinguishing the identity of bacterial isolates from patients and blood components. A patient with bacteremia can contaminate a PC in conjunction with a platelet transfusion. With AP-PCR, the PC could be ruled out as the cause of the posttransfusion sepsis.     

Bacteremia in childhood

Review of the bacteriology records of a University Hospital pediatric service for a 30-month period revealed 42 patients with Hemophilus influenzae type b bacteremia and 30 patients with Streptococcus pneumoniae bacteremia, all under age 10. Eighty-eight percent of the Hemophilus bacteremias and 7% of the pneumococcal bacteremias occurred in children less than 2 years of age. Hemophilus bacteremia was seen mot frequently in the first year of life, in contrast to pneumococcal bacteremia which was seen evenly throughout the first and second years of life. In all but one of the Hemophilus infections, a definite source of the bacteremia was apparent; these included CNS infection (58%), cellulitis (14%), and pneumonia (12%). In contrast, no obvious source was apparent in 37% of the pneumococcal bacteremias. When a focus for pneumococcal bacteremia was identified, otitis media and pneumonia were the most frequent diagnoses. Most of the occult pneumococcemias were transient; the results of repeat blood cultures before a treatment decision were helpful in determining the necessity for and duration of antibiotic therapy in those patients with no obvious source of infection.     

Epidemiology of pneumococcal serotype 6A and 6C among invasive and carriage isolates from Alaska, 1986–2009

We investigated serotype 6A/6C invasive pneumococcal disease (IPD) incidence, genetic diversity, and carriage before and after 7-valent pneumococcal conjugate vaccine (PCV7) introduction in Alaska. IPD cases (1986–2009) were identified through population-based laboratory surveillance. Isolates were initially serotyped by conventional methods, and 6C isolates were differentiated from 6A by polymerase chain reaction. Among invasive and carriage isolates initially typed as 6A, 35% and 50% were identified as 6C, respectively. IPD rates caused by serotype 6A or 6C among children <5 years did not change from the pre- to post-PCV7 period (P = 0.71 and P = 0.09, respectively). Multilocus sequence typing of IPD isolates revealed 28 sequence types. The proportion of serotype 6A carriage isolates decreased from 7.4% pre-PCV7 to 1.8% (P < 0.001) during 2008–2009; the proportion of serotype 6C carriage isolates increased from 3.0% to 8.4% (P = 0.004) among children <5 years. Continued surveillance is warranted to monitor changes in serotype distribution and prevalence.     

Streptococcus pneumoniae isolates with reduced susceptibility to ciprofloxacin in Spain: clonal diversity and appearance of ciprofloxacin-resistant epidemic clones

Analysis of the pulsed-field gel electrophoretic profiles of 82 pneumococcal isolates with reduced susceptibility to ciprofloxacin (RSC) and of 90 co-occurring susceptible isolates indicates a considerable genetic diversity among isolates with RCS and points to a close relation between the two groups. This finding suggests that pneumococci with RCS emerge through independent mutational events.