Acute lymphoblastic leukaemia patients treated with PEGasparaginase develop antibodies to PEG and the succinate linker.

Polyethylene glycol (PEG) conjugated asparaginase (PEGasparaginase) is essential for treatment of paediatric acute lymphoblastic leukaemia. We developed an assay identifying antibodies against the PEG-moiety, the linker and the drug itself in patients experiencing hypersensitivity reactions to PEGasparaginase. Eighteen patients treated according to the DCOG ALL-11 protocol, with a neutralizing hypersensitivity reaction to PEGasparaginase to the first PEGasparaginase doses in induction (12 patients) or during intensification after interruption of several months (6 patients) were included. ELISA was used to measure antibodies, coating with the succinimidyl succinate linker conjugated to BSA, PEGfilgrastim and Escherichia coli asparaginase, and using hydrolysed PEGasparaginase and mPEG5,000 for competition. Anti-PEG antibodies were detected in all patients (IgG 100%; IgM 67%) of whom 39% had anti-PEG antibodies exclusively. Pre-existing anti-PEG antibodies were also detected in patients who not previously received a PEGylated therapeutic (58% IgG; 21% IgM). Antibodies against the SS-linker were predominantly detected during induction (50% IgG; 42% IgM). Anti-asparaginase antibodies were detected in only 11% during induction but 94% during intensification. In conclusion, anti-PEG and anti-SS-linker antibodies predominantly play a role in the immunogenic response to PEGasparaginase during induction. Thus, switching to native E. coli asparaginase would be an option for adequate asparaginase treatment.

Within-Host Selection of Drug Resistance in a Mouse Model Reveals Dose-Dependent Selection of Atovaquone Resistance Mutations.

The evolutionary selection of malaria parasites within an individual host plays a critical role in the emergence of drug resistance. We have compared the selection of atovaquone resistance mutants in mouse models reflecting two different causes of failure of malaria treatment, an inadequate subtherapeutic dose and an incomplete therapeutic dose. The two models are based on cycles of insufficient treatment of Plasmodium berghei-infected mice: repeated inadequate treatment associated with a subtherapeutic dose (RIaT) (0.1 mg kg-1 of body weight) and repeated incomplete treatment with a therapeutic dose (RIcT) (14.4 mg kg-1 of body weight). The number of treatment cycles for the development of a stable resistance phenotype during RIaT was 2.00 ± 0.00 cycles (n = 9), which is not statistically different from that during RIcT (2.57 ± 0.85 cycles; combined n = 14; P = 0.0591). All mutations underlying atovaquone resistance selected by RIaT (M133I, T142N, and L144S) were found to be in the Qo1 (quinone binding 1) domain of the mitochondrial cytochrome b gene, in contrast to those selected by RIcT (Y268N/C, L271V, K272R, and V284F) in the Qo2 domain or its neighboring sixth transmembrane region. Exposure of mixed populations of resistant parasites from RIaT to the higher therapeutic dose of RIcT revealed further insights into the dynamics of within-host selection of resistance to antimalarial drugs. These results suggest that both inadequate subtherapeutic doses and incomplete therapeutic doses in malaria treatment pose similar threats to the emergence of drug resistance. RIcT and RIaT could be developed as useful tools to predict the potential emergence of resistance to newly introduced and less-understood antimalarials.

Within-Host Selection of Drug Resistance in a Mouse Model of Repeated Incomplete Malaria Treatment: Comparison between Atovaquone and Pyrimethamine.

The evolutionary selection of malaria parasites within individual hosts is an important factor in the emergence of drug resistance but is still not well understood. We have examined the selection process for drug resistance in the mouse malaria agent Plasmodium berghei and compared the dynamics of the selection for atovaquone and pyrimethamine. Resistance to these drugs has been shown to be associated with genetic lesions in the dihydrofolate reductase gene in the case of pyrimethamine and in the mitochondrial cytochrome b gene for atovaquone. A mouse malaria model for the selection of drug resistance, based on repeated incomplete treatment (RICT) with a therapeutic dose of antimalarial drugs, was established. The number of treatment cycles for the development of stable resistance to atovaquone (2.47 ± 0.70; n = 19) was found to be significantly lower than for pyrimethamine (5.44 ± 1.46; n = 16; P < 0.0001), even when the parental P. berghei Leiden strain was cloned prior to the resistance selection. Similar results were obtained with P. berghei Edinburgh. Mutational changes underlying the resistance were identified to be S110N in dihydrofolate reductase for pyrimethamine and Y268N, Y268C, Y268S, L271V-K272R, and G280D in cytochrome b for atovaquone. These results are consistent with the rate of mitochondrial DNA mutation being higher than that in the nucleus and suggest that mutation leading to pyrimethamine resistance is not a rare event.

Genogeography and Immune Epitope Characteristics of Hepatitis B Virus Genotype C Reveals Two Distinct Types: Asian and Papua-Pacific.

Distribution of hepatitis B virus (HBV) genotypes/subgenotypes is geographically and ethnologically specific. In the Indonesian archipelago, HBV genotype C (HBV/C) is prevalent with high genome variability, reflected by the presence of 13 of currently existing 16 subgenotypes. We investigated the association between HBV/C molecular characteristics with host ethnicity and geographical distribution by examining various subgenotypes of HBV/C isolates from the Asia and Pacific region, with further analysis on the immune epitope characteristics of the core and surface proteins. Phylogenetic tree was constructed based on complete HBV/C genome sequences from Asia and Pacific region, and genetic distance between isolates was also examined. HBV/C surface and core immune epitopes were analyzed and grouped by comparing the amino acid residue characteristics and geographical origins. Based on phylogenetic tree and geographical origins of isolates, two major groups of HBV/C isolates--East-Southeast Asia and Papua-Pacific--were identified. Analysis of core and surface immune epitopes supported these findings with several amino acid substitutions distinguishing the East-Southeast Asia isolates from the Papua-Pacific isolates. A west-to-east gradient of HBsAg subtype distribution was observed with adrq+ prominent in the East and Southeast Asia and adrq- in the Pacific, with several adrq-indeterminate subtypes observed in Papua and Papua New Guinea (PNG). This study indicates that HBV/C isolates can be classified into two types, the Asian and the Papua-Pacific, based on the virus genome diversity, immune epitope characteristics, and geographical distribution, with Papua and PNG as the molecular evolutionary admixture region in the switching from adrq+ to adrq-.

Differences in the reporting rates of serious allergic adverse events from intravenous iron by country and population.

BACKGROUND: Previous studies have compared rates of adverse events between intravenous (IV) iron preparations; however, there has been no comparison of adverse event rates by country and population. OBJECTIVES: To compare rates of adverse events to IV iron products by country and population. METHODS: All adverse events reported from 18 countries from January 1, 2003 to June 30, 2009 were obtained for iron dextran (ID), iron sucrose (IS), IS similars (ISS), and sodium ferric gluconate (FG). Rates of all adverse events and serious adverse events (anaphylaxis plus other serious allergic reactions) were calculated as number of events per gram of iron sold (gFe) per million inhabitants (mil) × 10-3. Odds ratios (ORs) were calculated for the risks of adverse events between products. RESULTS: Iron use ranged from 1 gFe/mil (Poland) to 48,674 gFe/mil (Italy). Rates of all adverse events (reports/gFe/mil × 10-3) varied: for IS, it ranged from 0 (Poland, Austria, Czech Republic) to 1,222 (Ireland); for FG, from 3.3 (Czech Republic) to 183.6 (United States); for ID, from 0.9 (Turkey) to 46,875 (Switzerland). There were no reports of adverse events in ISS. In a subset of countries that used 2 or more iron products and had more than 1 serious adverse event, rates (reports/gFe/mil × 10-3) of all adverse events and serious adverse events were lowest for IS (39.8 and 1.7), intermediate for FG (54.8 and 4.5), and greatest for ID (337.7 and 20.5). IS had lower risks for all adverse events (OR, 0.63; P<.0001) and serious adverse events (OR, 0.31; P=.001) versus FG, and for all adverse events (OR, 0.13; P<.0001) and serious adverse events (OR, 0.07; P<.0001) versus ID. FG had lower risks for all adverse events (OR, 0.20; P<.0001) and serious adverse events (OR, 0.24; P<.0001) versus ID. CONCLUSIONS: Considerable international variation existed in the extent and choice of iron product and adverse event reporting, suggesting under-reporting in some instances. Clinicians should appreciate the differential risks between available products, and should critically review local reporting practices.

Differences in spontaneously reported hypersensitivity and serious adverse events for intravenous iron preparations: comparison of Europe and North America.

Spontaneously-reported rates of adverse events (AEs) of intravenous (i.v.) iron products have not been compared since 2007. AEs in Europe (Eur) and North America (NA) have never been compared. New products have been marketed and many changes in prescribing habits have occurred since then, and the effect on AEs reporting is unclear. It was hypothesized that changing practices for i.v. iron products has caused changes in the rates of serious AEs and large differences exist between Eur and NA. Rates of AEs for three i.v. iron preparations (iron sucrose [IS], ferric gluconate [FG] and high and low MW iron dextran [HMWID, LMWID]) were compared by product and continent from January 1, 2003 to June 30, 2009 for selected countries in Eur and NA, using the Uppsala Monitoring Center's database. Rates of total, anaphylaxis and other serious allergic AEs were calculated as number of AEs divided by i.v. iron sales standardized to 100 mg dose equivalents (DEq) of iron. Quarterly sales (millions of 100 mg DEq of iron) increased from the first quarter 2003 to the end of the second quarter of 2009 by 1% for FG, 16% for IS and 2% for ID. Total AEs for NA plus Eur were similar for FG and IS, but total AEs were 6- to 7-fold higher for ID. Rates of anaphylaxis were 6- to 11-fold higher in Eur plus NA combined for ID than for IS or FG. In NA, there were substantially higher reports for total, anaphylaxis and other serious allergic AEs with FG compared to IS, whereas the reverse was the case in Eur. Odds ratios (OR) showed higher risks of anaphylaxis with FG in NA vs. Eur (OR = 4.40, P < 0.0001) and lower risks with IS (OR = 0.24, P < 0.0001). Odds of anaphylaxis with LMWID in Eur vs. FG and IS were 42.08 and 16.92 (both P < 0.0001), respectively. In NA, odds of anaphylaxis with ID vs. FG and IS were 2.36 and 17.73 (both P < 0.0001), respectively. Differences between NA and Eur may be related to varied treatment practices. ID had the highest rates of all types of AEs, and IS and FG had a continued trend for lowest rates of AEs.

Neutralization of Neisseria meningitidis outer membrane vesicles.

INTRODUCTION: We aimed to determine the neutralization of Neisseria meningitidis outer membrane vesicles (blebs) by humoral and cellular elements of whole blood. METHODS: The interaction of FITC-labeled blebs with monocytes was studied by spectrofluorometry. Blebs are able to induce an oxidative burst in neutrophils, and we evaluated the inhibitory effect of plasma on this process. RESULTS: Human plasma reduced the priming activity of blebs containing 1-3 ng/ml lipopolysaccharide (LPS) by 50-60% and bactericidal permeability increasing protein (BPI) reduced priming to background levels. A complete neutralization of LPS and blebs by plasma and BPI was measured using the limulus amebocyte lysate (LAL) assay. Furthermore, only 3% of blebs were cell-associated, while the remainder were in the supernatant. CONCLUSIONS: Plasma and BPI are able to neutralize blebs, with phagocytosis playing only a minor role. As such, we conclude that blebs do not behave like particles but more like free LPS.

Colonization and transmission of meticillin-susceptible and meticillin-resistant Staphylococcus aureus in a murine nasal colonization model.

Nasal colonization by Staphylococcus aureus is an important risk factor for the development of a nosocomial infection. Acquisition of nasal colonization by S. aureus increases mortality in hospitalized patients, but little is known about the transmission dynamics of S. aureus. To study S. aureus transmission, colonization and colonization persistence, we developed a murine transmission model. In 20 cages, 2 out of 10 mice were nasally inoculated (at 5×10(8) c.f.u. per mouse) with either meticillin-susceptible S. aureus (MSSA) (10 cages) or meticillin-resistant S. aureus (MRSA) (10 cages). On days 5, 15, 25 and 40, all mice in a cage were swabbed or sacrificed and nasal colonization and c.f.u. were determined in all 10 mice by nasal dissection or by nasal swab. Spread and subsequent stable colonization by both MSSA and MRSA from colonized to uncolonized mice within a cage was seen. At day 5, an increased number of colonized mice were observed in the MSSA group compared to the MRSA group (P = 0.003). On day 40, the mean number of c.f.u. per mouse was higher for MRSA than for MSSA (P = 0.06). Faecal-oral transmission was shown to be a possibly important transmission route in this model. These results suggest a more rapid spread of MSSA compared to MRSA. However, MRSA shows a more stable nasal colonization after a longer period of time.

Ethnogeographical structure of hepatitis B virus genotype distribution in Indonesia and discovery of a new subgenotype, B9.

The distribution of hepatitis B virus (HBV) in the populations of island Southeast Asia is of medical and anthropological interest and is associated with an unusually high genetic diversity. This study examined the association of this HBV genetic diversity with the ethnogeography of the populations of the Indonesian archipelago. Whole genome analysis of 21 HBV isolates from East Nusa Tenggara and Papua revealed two recently reported HBV/B subgenotypes unique to the former, B7 (7 isolates) and B8 (5 isolates), and uncovered a further novel subgenotype designated B9 (4 isolates). Further isolates were collected from 419 individuals with defined ethnic backgrounds representing 40 populations. HBV/B was predominant in Austronesian-language-speaking populations, whereas HBV/C was the major genotype in Papua and Papua-influenced populations of Moluccas; HBV/B3 was the predominant subgenotype in the western half of the archipelago (speakers of the Western Malayo-Polynesian [WMP] branch of Austronesian languages), whereas B7, B8 and B9 were specific to Nusa Tenggara (Central Malayo-Polynesian (CMP)). The result provides the first direct evidence that the distribution of HBV genotypes/subgenotypes in the Indonesian archipelago is related to the ethnic origin of its populations and suggests that the HBV distribution is associated with the ancient migratory events in the peopling of the archipelago.

Evolution in quantum leaps: multiple combinatorial transfers of HPI and other genetic modules in Enterobacteriaceae.

Horizontal gene transfer is a key step in the evolution of Enterobacteriaceae. By acquiring virulence determinants of foreign origin, commensals can evolve into pathogens. In Enterobacteriaceae, horizontal transfer of these virulence determinants is largely dependent on transfer by plasmids, phages, genomic islands (GIs) and genomic modules (GMs). The High Pathogenicity Island (HPI) is a GI encoding virulence genes that can be transferred between different Enterobacteriaceae. We investigated the HPI because it was present in an Enterobacter hormaechei outbreak strain (EHOS). Genome sequence analysis showed that the EHOS contained an integration site for mobile elements and harbored two GIs and three putative GMs, including a new variant of the HPI (HPI-ICEEh1). We demonstrate, for the first time, that combinatorial transfers of GIs and GMs between Enterobacter cloacae complex isolates must have occurred. Furthermore, the excision and circularization of several combinations of the GIs and GMs was demonstrated. Because of its flexibility, the multiple integration site of mobile DNA can be considered an integration hotspot (IHS) that increases the genomic plasticity of the bacterium. Multiple combinatorial transfers of diverse combinations of the HPI and other genomic elements among Enterobacteriaceae may accelerate the generation of new pathogenic strains.

Yersiniabactin reduces the respiratory oxidative stress response of innate immune cells.

Enterobacteriaceae that contain the High Pathogenicity Island (HPI), which encodes the siderophore yersiniabactin, display increased virulence. This increased virulence may be explained by the increased iron scavenging of the bacteria, which would both enhance bacterial growth and limit the availability of iron to cells of the innate immune system, which require iron to catalyze the Haber-Weiss reaction that produces hydroxyl radicals. In this study, we show that yersiniabactin increases bacterial growth when iron-saturated lactoferrin is the main iron source. This suggests that yersiniabactin provides bacteria with additional iron from saturated lactoferrin during infection. Furthermore, the production of ROS by polymorphonuclear leukocytes, monocytes, and a mouse macrophage cell line is blocked by yersiniabactin, as yersiniabactin reduces iron availability to the cells. Importantly, iron functions as a catalyst during the Haber-Weiss reaction, which generates hydroxyl radicals. While the physiologic role of the Haber-Weiss reaction in the production of hydroxyl radicals has been controversial, the siderophores yersiniabactin, aerobactin, and deferoxamine and the iron-chelator deferiprone also reduce ROS production in activated innate immune cells. This suggests that this reaction takes place under physiological conditions. Of the tested iron chelators, yersiniabactin was the most effective in reducing the ROS production in the tested innate immune cells. The likely decreased bacterial killing by innate immune cells resulting from the reduced production of hydroxyl radicals may explain why the HPI-containing Enterobacteriaceae are more virulent. This model centered on the reduced killing capacity of innate immune cells, which is indirectly caused by yersiniabactin, is in agreement with the observation that the highly pathogenic group of Yersinia is more lethal than the weakly pathogenic and the non-pathogenic group.

Identification of resistance and virulence factors in an epidemic Enterobacter hormaechei outbreak strain.

Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment.

Acquisition and cross-transmission of Staphylococcus aureus in European intensive care units.

OBJECTIVE: To study the acquisition and cross-transmission of Staphylococcus aureus in different intensive care units (ICUs). METHODS: We performed a multicenter cohort study. Six ICUs in 6 countries participated. During a 3-month period at each ICU, all patients had nasal and perineal swab specimens obtained at ICU admission and during their stay. All S. aureus isolates that were collected were genotyped by spa typing and multilocus variable-number tandem-repeat analysis typing for cross-transmission analysis. A total of 629 patients were admitted to ICUs, and 224 of these patients were found to be colonized with S. aureus at least once during ICU stay (22% were found to be colonized with methicillin-resistant S. aureus [MRSA]). A total of 316 patients who had test results negative for S. aureus at ICU admission and had at least 1 follow-up swab sample obtained for culture were eligible for acquisition analysis. RESULTS: A total of 45 patients acquired S. aureus during ICU stay (31 acquired methicillin-susceptible S. aureus [MSSA], and 14 acquired MRSA). Several factors that were believed to affect the rate of acquisition of S. aureus were analyzed in univariate and multivariate analyses, including the amount of hand disinfectant used, colonization pressure, number of beds per nurse, antibiotic use, length of stay, and ICU setting (private room versus open ICU treatment). Greater colonization pressure and a greater number of beds per nurse correlated with a higher rate of acquisition for both MSSA and MRSA. The type of ICU setting was related to MRSA acquisition only, and the amount of hand disinfectant used was related to MSSA acquisition only. In 18 (40%) of the cases of S. aureus acquisition, cross-transmission from another patient was possible. CONCLUSIONS: Colonization pressure, the number of beds per nurse, and the treatment of all patients in private rooms correlated with the number of S. aureus acquisitions on an ICU. The amount of hand disinfectant used was correlated with the number of cases of MSSA acquisition but not with the number of cases of MRSA acquisition. The number of cases of patient-to-patient cross-transmission was comparable for MSSA and MRSA.

Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells.

BACKGROUND: Mannose binding lectin (MBL) is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro. RESULTS: High avidity binding was observed between MBL and C. albicans and C. parapsilosis. Addition of MBL to MBL deficient serum increased the deposition of C4 and C3b and enhanced the uptake of C. albicans, C. parapsilosis and acapsular C. neoformans by polymorphonuclear cells (PMNs). Compared to other microorganisms, such as Escherichia coli, Staphylococcus aureus and Cryptococcus neoformans, C. parapsilosis and Candida albicans were potent activators of the lectin pathway. CONCLUSION: Our results suggest that MBL plays a crucial role in the innate immunity against infections caused by yeast by increasing uptake by PMN.

Genomic diversity within the Enterobacter cloacae complex.

BACKGROUND: Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome. METHODOLOGY/PRINCIPAL FINDINGS: This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogeneous. Genetic markers were identified that could discriminate between the two clades and cluster 1. CONCLUSIONS/SIGNIFICANCE: Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.

Novel multiple-locus variable-number tandem-repeat analysis method for rapid molecular typing of human Staphylococcus aureus.

We evaluated the use of a novel multiple-locus variable-number tandem-repeat analysis (MLVA) method for typing of human Staphylococcus aureus. For a total of 150 clinical isolates, MLVA demonstrated the highest discriminatory power. MLVA correctly assigned isolates to outbreaks or identified isolates as unlinked. MLVA is a rapid and simple method for the epidemiological typing of S. aureus.

Quantifying the relationship between Staphylococcus aureus bacteremia and S. aureus bacteriuria: a retrospective analysis in a tertiary care hospital.

In this retrospective cohort study, patients who had Staphylococcus aureus bacteremia but who lacked signs and symptoms of urinary tract infection due to S. aureus and who did not have an indwelling urinary catheter had a likelihood of S. aureus bacteriuria of 2.5% (2 of 79 patients). Therefore, we strongly question the theory that S. aureus bacteremia causes S. aureus bacteriuria.

Dynamics in prophage content of invasive and noninvasive M1 and M28 Streptococcus pyogenes isolates in The Netherlands from 1959 to 1996.

Invasive group A streptococcal (GAS) disease re-emerged in The Netherlands in the late 1980s. To seek an explanation for this resurgence, the genetic compositions of 22 M1 and 19 M28 GAS strains isolated in The Netherlands between 1960s and the mid-1990s were analyzed by using a mixed-genome DNA microarray. During this four-decade period, M1 and especially M28 strains acquired prophages on at least eight occasions. All prophages carried a superantigen (speA2, speC, speK) or a streptodornase (sdaD2, sdn), both associated with invasive GAS disease. Invasive and noninvasive GAS strains did not differ in prophage acquisition, suggesting that there was an overall increase in the pathogenicity of M1 and M28 strains over the last four decades rather than emergence of hypervirulent subclones. The increased overall pathogenic potential may have contributed to the reemergence of invasive GAS disease in The Netherlands.

Determination of the relationship between group A streptococcal genome content, M type, and toxic shock syndrome by a mixed genome microarray.

Group A streptococci (GAS), or Streptococcus pyogenes, are associated with a remarkable variety of diseases, ranging from superficial infections to life-threatening diseases such as toxic-shock-like syndrome (TSS). GAS strains belonging to M types M1 and M3 are associated with TSS. This study aims to obtain insight into the gene profiles underlying different M types and disease manifestations. Genomic differences between 76 clinically well characterized GAS strains collected in The Netherlands were examined using a mixed-genome microarray. Inter-M-type genomic differences clearly outweighed intra-M-type genome variation. Phages were major contributors to observed genome diversification. We identified four novel genes, including two genes encoding fibronectin-binding-like proteins, which are highly specific to a subset of M types and thus may contribute to M-type-associated disease manifestations. All M12 strains were characterized by the unique absence of the citrate lyase complex and reduced growth under hypoxic, nutrient-deprived conditions. Furthermore, six virulence factors, including genes encoding a complement-inhibiting protein (sic), an exotoxin (speA), iron(III) binding factor, collagen binding factor (cpa), and fibrinogen binding factor (prt2-like), were unique to M1 and/or M3 strains. These virulence factors may contribute to the potential of these strains to cause TSS. Finally, in contrast to M-type-specific virulence profiles, we did not identify a common virulence profile among strains associated with TSS irrespective of their M type.

Failure to control an outbreak of qnrA1-positive multidrug-resistant Enterobacter cloacae infection despite adequate implementation of recommended infection control measures.

A large outbreak with an aminoglycoside-resistant Enterobacter cloacae (AREC) clone occurred at the University Medical Center Utrecht beginning in 2001 and continued up through the time that this study was completed. This clone (genotype I) contains a conjugative R plasmid carrying the qnrA1, bla(CTX-M-9), and aadB genes, encoding resistance to quinolones, extended-spectrum beta-lactamases, and aminoglycosides, respectively. The aim of this study was to determine whether this clone was more transmissible than other AREC strains. Therefore, the dissemination of this genotype and of other E. cloacae strains was studied. In addition, infection control measures taken were evaluated. Pulsed-field gel electrophoresis analysis divided the 191 AREC strains into 42 different genotypes, of which 5 (12%) involved at least three patients. Aside from this outbreak (133 patients), only two other small outbreaks occurred, showing that the infection control measures were successful for all strains but one. Among 324 aminoglycoside-susceptible E. cloacae strains, 34/166 (20%) genotypes were identified from at least three patients, but only 4 involved small outbreaks. The outbreak strain was also detected in 11 of 15 other Dutch hospitals and caused outbreaks in at least 4. Evaluation of infection control measures showed that the outbreak strain disseminated throughout the hospital despite adequate implementation of internationally accepted guidelines on the control of multidrug-resistant Enterobacteriaceae (MRE). In conclusion, some MRE strains are better able to spread than others, and these strains may not be controlled by the current infection control guidelines. Strategies to identify such strains in an early phase and adapted guidelines for such "superbugs" are needed to prevent these clones from becoming endemic.