Jan Verhoef

Multilocus Sequence Typing of Klebsiella pneumoniae Nosocomial Isolates

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

Emerging Importance of Multidrug-Resistant Acinetobacter Species and Stenotrophomonas maltophilia as Pathogens in Seriously Ill Patients: Geographic Patterns, Epidemiological Features, and Trends in the SENTRY Antimicrobial Surveillance Program (1997–1999)

As part of the SENTRY Antimicrobial Surveillance Program, a total of 1078 Acinetobacter species and 842 Stenotrophomonas maltophilia isolates were collected between January 1997 and December 1999 from 5 geographic regions (Canada, the United States, Latin America, Europe, and the Asia-Pacific). The frequency of infections (by geographic region and body site), including those due to imipenem-resistant Acinetobacter species and trimethoprim-sulfamethoxazole (TMP-SMZ)-resistant S. maltophilia, was evaluated. The possibility of seasonal variations in bloodstream infections caused by Acinetobacter species was studied, as was the activity of several therapeutic antimicrobials against all strains. Acinetobacter species and S. maltophilia were most frequently associated with pulmonary infections, independent of the region evaluated. In contrast, patterns of antimicrobial resistance markedly varied among distinct geographic regions, especially for nosocomial isolates. Although the carbapenems were the most active antimicrobials against Acinetobacter species, nearly 11.0% of the nosocomial isolates were resistant to this drug group in both regions. TMP-SMZ, ticarcillin-clavulanic acid, gatifloxacin, and trovafloxacin were the only agents with consistent therapeutic activity against S. maltophilia isolates. Rates of resistance to TMP-SMZ ranged from 2% in Canada and Latin America to 10% in Europe. The geographic differences in resistance patterns among Acinetobacter species and S. maltophilia isolates observed in this study emphasize the importance of local surveillance in determining the most adequate therapy for acinetobacter and S. maltophilia infections and the possible clonal, epidemic nature of occurrence.

Molecular typing of methicillin-resistantStaphylococcus aureus on the basis of protein A gene polymorphism

The polymorphic X-region of the protein A gene (spa) was used for molecular typing of methicillin-resistantStaphylococcus aureus (MRSA) strains. The X-region is characterized by a variable number (between 3 and 15) of small repeats. DNA sequencing of MRSA strains revealed 25 distinct repeats. Analysis of MRSA strains grown in vitro and in vivo revealed that the X-region was sufficiently stable for epidemiologic typing of MRSA strains.Spa typing of MRSA strains was compared to phage typing and, in general, concordance was found between the two methods. However,spa typing was more sensitive, allowing differentiation of strains within a particular phage type. Results obtained withspa typing suggest that hospital outbreaks may be caused by two or more MRSA strains.Spa typing may be an important tool in unravelling the spread of MRSA strains within and between hospitals.

Antimicrobial Susceptibility and Frequency of Occurrence of Clinical Blood Isolates in Europe from the SENTRY Antimicrobial Surveillance Program, 1997 and 1998

As part of the European arm of the SENTRY Antimicrobial Surveillance Program, 25 European university hospitals referred 9613 blood isolates for in vitro testing against >20 antimicrobial agents. Escherichia coli, Staphylococcus aureus, coagulase-negative Staphylococcus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the 5 most frequent isolates and accounted for two-thirds of all referrals, with minor regional variation. Of these, approximately 0.36% of E. coli and 16.7% of K. pneumoniae isolates proved to be potential extended-spectrum beta-lactamase producers, and their incidence clearly varied regionally. Quinolone resistance was detected among gram-negative species; in particular, P. aeruginosa and Acinetobacter species. Considerable regional variation was observed in the incidences of methicillin resistance in S. aureus and penicillin resistance in Streptococcus pneumoniae. The incidence of vancomycin resistance in enterococci was relatively low overall and primarily associated with Enterococcus faecium. However, extrapolation of these data to smaller and nonteaching hospitals should be undertaken with caution, since resistance rates may be lower in these facilities.

Epidemiology and Susceptibility of 3,051 Staphylococcus aureus Isolates from 25 University Hospitals Participating in the European SENTRY Study

ABSTRACT A total of 3,051 methicillin-susceptible Staphylococcus aureus (MSSA) isolates and methicillin-resistant S. aureus (MRSA) isolates in Europe were compared. MRSA isolates constituted 25% of all isolates and were more prevalent in southern Europe. MRSA isolates appeared to be more prevalent in intensive care units than in outpatient departments. Only a small minority of MSSA isolates were multidrug resistant, whereas the majority of MRSA isolates were multidrug resistant.

International surveillance of blood stream infections due to Candida species in the European SENTRY program: species distribution and antifungal susceptibility including the investigational triazole and echinocandin agents

The SENTRY Antimicrobial Surveillance Program, an international study of blood stream infections (BSIs), detected 170 episodes of candidemia in 20 European medical centers (13 nations) between January and December, 1997. Twenty-three percent of the candidal BSI occurred in patients hospitalized in an intensive care unit, 21% in patients in an internal medicine service, 13% in patients in a surgical service, and 9% in patients in an oncology service. Overall, 53% of the BSI were attributable to Candida albicans followed in prevalence by C. parapsilosis (21%), C. glabrata (12%), C. tropicalis (6%), C. famata (2%), C. krusei (1%), and C. inconspicua (1%). As observed previously in Canada and Latin America, C. parapsilosis and not C. glabrata, was the most common non-albicans species causing yeast BSI in Europe. The proportion of these candidemias attributable to C. albicans varied widely from 0-100% among the 20 European centers. Among the different species of Candida, resistance to fluconazole (MIC, > or = 64 micrograms/mL) and itraconazole (MIC, > or = 1.0 microgram/mL) was observed with C. glabrata and C. krusei and was observed more rarely among other species (e.g., C. inconspicua). Isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. guilliermondii were all highly susceptible to both fluconazole and itraconazole. Furthermore, the investigational triazoles (BMS-207147, Sch 56592, and voriconazole) and an echinocandin (MK-0991) all demonstrated potent in vitro activity (MIC90s, 0.5, 0.5, 1.0, and 2.0 micrograms/mL, respectively) against these isolates. Continued surveillance at an international level will be important to monitor trends in species distribution and antifungal susceptibility among invasive strains of Candida.

Prevention of colonization and infection in critically ill patients: a prospective randomized study.

In a prospective randomized study to determine whether prevention of colonization of Gram-negative bacteria results in prevention of Gram-negative bacterial infections, 96 intensive care patients were randomly allocated into a control group and a study group. The study group received oral nonabsorbable antimicrobial agents (i.e., tobramycin, amphotericin B, and polymyxin E) in addition to parenteral antibiotics. Colonization with Gram-negative microorganisms in the oropharynx, and respiratory and digestive tracts increased in the control group during their stay, while the study group did not tend to colonize with Gram-negative bacteria. In the control group, 107 nosocomial infections were diagnosed, vs. 42 nosocomial infections in the study group. Nosocomial infections caused by Gram-negative bacteria were significantly less frequent in the study group. Mortality due to an acquired infection was significantly less frequent in the study group. We conclude that colonization, infection, and subsequent mortality by nosocomial Gram-negative bacteria can be prevented by a regime of topically applied nonabsorbable antibiotics.

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Salpha rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa-IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Salpha rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Salpha rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Salpha rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Salpha rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.

Lipoprotein metabolism in patients with severe sepsis

ObjectiveLipoproteins have been implicated to play a role in innate immunity. Changes in lipoprotein levels have been reported in a variety of inflammatory disorders. Not much is known about lipoprotein metabolism in patients with severe sepsis. We conducted an ancillary study in a multiple-center phase III sepsis trial to investigate the dynamics of plasma lipoproteins in patients with severe sepsis. DesignProspective analysis in patients meeting criteria for severe sepsis as part of a multiple-center sepsis study (KyberSept) with antithrombin III (Kybernin P). SettingUniversity hospital intensive care unit. PatientsSeventeen patients were included in the study. InterventionsRandomized patients received a loading dose of 6000 IU of antithrombin III (Kybernin P) or placebo followed by a 96-hr continuous infusion of 250 IU/hr antithrombin III (Kybernin P) or placebo. In each patient, serial blood samples for total cholesterol, lipoprotein cholesterol, triglycerides, apolipoprotein A-1, apolipoprotein B, and C-reactive protein determination as well as clinical data were collected over 28 days. Measurements and Main ResultsPlasma cholesterol levels rapidly decreased from 2.67 ± 2.02 mmol/L on day 0 to a nadir of 1.41 ± 0.70 mmol/L on day 3, followed by a slow increase to 4.18 ± 1.94 mmol/L on day 28. High-density lipoprotein (HDL) cholesterol concentrations decreased rapidly from 0.84 ± 0.92 mmol/L to a nadir of 0.42 ± 0.35 mmol/L on day 3, to show a slow increase during the following 4 wks to 0.84 ± 0.42 mmol/L. The low-density lipoprotein (LDL) cholesterol concentrations were already low (0.94 ± 0.81 mmol/L) at study entry, to show a progressive increase to subnormal values (2.01 ± 0.94 mmol/L) at 4 wks. Nadir and recovery lipoprotein concentrations were significantly different (paired Student’s t-test, p < .05). A significant correlation was found between HDL cholesterol and apolipoprotein A-1 (r = .714, p < .05) and between LDL cholesterol and apolipoprotein B (r = .733, p < .05). There was no statistical difference in lipoprotein concentrations either between survivors and nonsurvivors or between patients receiving antithrombin III or placebo.Serum amyloid A was a major apoprotein (45%) in HDL at the start of the sepsis and was slowly replaced by apolipoprotein A-1 during recovery. A positive correlation was found between plasma C-reactive protein concentrations and serum amyloid A concentrations in HDL (r = .684, p < .05). No other relevant correlations were found between inflammatory and lipoprotein parameters. ConclusionsIn patients with severe sepsis, lipoprotein concentrations rapidly change and can be reduced to 50% of recovery concentrations. The pattern of early rapid decline is found primarily in the HDL and a slow recovery in both HDL and LDL fractions. The correlation between apolipoprotein and lipoprotein cholesterol concentrations suggests a decline in lipoprotein particles. During severe sepsis, HDL is shifted to acute phase HDL, which is enriched in serum amyloid A and depleted of cholesterol and apolipoprotein A-1. Lipoprotein concentrations are unable to discriminate between survivors and nonsurvivors.

Infection Prophylaxis in Acute Leukemia: A Comparison of Ciprofloxacin with Trimethoprim-Sulfamethoxazole and Colistin

Fifty-six patients receiving remission induction treatment for acute leukemia were studied in a randomized trial comparing ciprofloxacin with trimethoprim-sulfamethoxazole plus colistin for prevention of infections. Both groups received amphotericin B for antifungal prophylaxis. Six major infections occurred in 28 patients receiving ciprofloxacin, and 11 major infections occurred in 28 patients receiving trimethoprim-sulfamethoxazole plus colistin. No infections caused by gram-negative bacilli were seen in the ciprofloxacin group (p less than 0.02). Ciprofloxacin prevented colonization with resistant gram-negative bacilli, but 12 resistant colonizing strains were isolated from 10 patients receiving trimethoprim-sulfamethoxazole plus colistin (p less than 0.01). Ciprofloxacin was better tolerated: 23 of 28 patients were highly compliant to the drug, compared with 15 of 28 patients in the trimethoprim-sulfamethoxazole group (p less than 0.05). These results suggest that ciprofloxacin is a promising drug for the prevention of infection in patients with granulocytopenia.