Invasive mould infections: a multi-disciplinary update.

Systemic fungal infections remain a significant cause of mortality in neutropenic and immunocompromised patients, despite advances in their diagnosis and treatment. The incidence of such infections is rising due to the use of intensive chemotherapy regimens in patients with solid tumours or haematological cancers, the increasing numbers of allogeneic haematopoietic stem cell and solid organ transplants, and the use of potent immunosuppressive therapy in patients with autoimmune disorders. In addition, the epidemiology of systemic fungal infections is changing, with atypical species such as Aspergillus terreus and zygomycetes becoming more common. Treatment has traditionally focused on empirical therapy, but targeted pre-emptive therapy in high-risk patients and prophylactic antifungal treatment are increasingly being adopted. New treatments, including lipid formulations of amphotericin B, second-generation broad-spectrum azoles, and echinocandins, offer effective antifungal activity with improved tolerability compared with older agents; the potential impact of these treatments is reflected in their inclusion in current treatment and prophylaxis guidelines. New treatment strategies, such as aerosolized lipid formulations of amphotericin B, may also reduce the burden of mortality associated with systemic fungal infections. The challenge is to identify ways of coupling potentially effective treatments with early and reliable identification of patients at highest risk of infection.

Molecular evidence of interhuman transmission of Pneumocystis pneumonia among renal transplant recipients hospitalized with HIV-infected patients.

Ten Pneumocystis jirovecii pneumonia (PCP) cases were diagnosed in renal transplant recipients (RTRs) during a 3-year period. Nosocomial transmission from HIV-positive patients with PCP was suspected because these patients shared the same hospital building, were not isolated, and were receiving suboptimal anti-PCP prophylaxis or none. P. jirovecii organisms were typed with the multitarget polymerase chain reaction-single-strand conformation polymorphism method. Among the 45 patients with PCP hospitalized during the 3-year period, 8 RTRs and 6 HIV-infected patients may have encountered at least 1 patient with active PCP within the 3 months before the diagnosis of their own PCP episode. In six instances (five RTRs, one HIV-infected patient), the patients harbored the same P. jirovecii molecular type as that found in the encountered PCP patients. The data suggest that part of the PCP cases observed in this building, particularly those observed in RTRs, were related to nosocomial interhuman transmission.

Usefulness of double locus sequence typing (DLST) for regional and international epidemiological surveillance of methicilin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. To differentiate reliably among S. aureus isolates, we recently developed double locus sequence typing (DLST) based on the analysis of partial sequences of clfB and spa genes. In the present study, we evaluated the usefulness of DLST for epidemiological investigations of MRSA by routinely typing 1242 strains isolated in Western Switzerland. Additionally, particular local and international collections were typed by pulsed field gel electrophoresis (PFGE) and DLST to check the compatibility of DLST with the results obtained by PFGE, and for international comparisons. Using DLST, we identified the major MRSA clones of Western Switzerland, and demonstrated the close relationship between local and international clones. The congruence of 88% between the major PFGE and DLST clones indicated that our results obtained by DLST were compatible with earlier results obtained by PFGE. DLST could thus easily be incorporated in a routine surveillance procedure. In addition, the unambiguous definition of DLST types makes this method more suitable than PFGE for long-term epidemiological surveillance. Finally, the comparison of the results obtained by DLST, multilocus sequence typing, PFGE, Staphylococcal cassette chromosome mec typing and the detection of Panton-Valentine leukocidin genes indicated that no typing scheme should be used on its own. It is only the combination of data from different methods that gives the best chance of describing precisely the epidemiology and phylogeny of MRSA.

Quantitative antibiogram as a typing method for the prospective epidemiological surveillance and control of MRSA: comparison with molecular typing.

OBJECTIVE: Evaluation of the quantitative antibiogram as an epidemiological tool for the prospective typing of methicillin-resistant Staphylococcus aureus (MRSA), and comparison with ribotyping. METHODS: The method is based on the multivariate analysis of inhibition zone diameters of antibiotics in disk diffusion tests. Five antibiotics were used (erythromycin, clindamycin, cotrimoxazole, gentamicin, and ciprofloxacin). Ribotyping was performed using seven restriction enzymes (EcoRV, HindIII, KpnI, PstI, EcoRI, SfuI, and BamHI). SETTING: 1,000-bed tertiary university medical center. RESULTS: During a 1-year period, 31 patients were found to be infected or colonized with MRSA. Cluster analysis of antibiogram data showed nine distinct antibiotypes. Four antibiotypes were isolated from multiple patients (2, 4, 7, and 13, respectively). Five additional antibiotypes were isolated from the remaining five patients. When analyzed with respect to the epidemiological data, the method was found to be equivalent to ribotyping. Among 206 staff members who were screened, six were carriers of MRSA. Both typing methods identified concordant of MRSA types in staff members and in the patients under their care. CONCLUSIONS: The quantitative antibiogram was found to be equivalent to ribotyping as an epidemiological tool for typing of MRSA in our setting. Thus, this simple, rapid, and readily available method appears to be suitable for the prospective surveillance and control of MRSA for hospitals that do not have molecular typing facilities and in which MRSA isolates are not uniformly resistant or susceptible to the antibiotics tested.

Burkholderia sartisoli sp. nov., isolated from a polycyclic aromatic hydrocarbon-contaminated soil.

A Gram-negative, rod-shaped, aerobic bacterium, designated strain RP007(T), was isolated from a polycyclic aromatic hydrocarbon-contaminated soil in New Zealand. Two additional strains were recovered from a compost heap in Belgium (LMG 18808) and from the rhizosphere of maize in the Netherlands (LMG 24204). The three strains had virtually identical 16S rRNA gene sequences and whole-cell protein profiles, and they were identified as members of the genus Burkholderia, with Burkholderia phenazinium as their closest relative. Strain RP007(T) had a DNA G+C content of 63.5 mol% and could be distinguished from B. phenazinium based on a range of biochemical characteristics. Strain RP007(T) showed levels of DNA-DNA relatedness towards the type strain of B. phenazinium and those of other recognized Burkholderia species of less than 30 %. The results of 16S rRNA gene sequence analysis, DNA-DNA hybridization experiments and physiological and biochemical tests allowed the differentiation of strain RP007(T) from all recognized species of the genus Burkholderia. Strains RP007(T), LMG 18808 and LMG 24204 are therefore considered to represent a single novel species of the genus Burkholderia, for which the name Burkholderia sartisoli sp. nov. is proposed. The type strain is RP007(T) (=LMG 24000(T) =CCUG 53604(T) =ICMP 13529(T)).

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication.

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection.

Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.

The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.

Ecological temporal stability of Staphylococcus aureus nasal carriage.

We described the colonization dynamics of Staphylococcus aureus in a group of 266 healthy carriers over a period of approximately 1 year. We used precise genotyping methods, i.e., amplified fragment length polymorphism (AFLP), spa typing, and double-locus sequence typing (DLST), to detect changes in strain identity. Strain change took place rather rarely: out of 89 carriers who had initially been colonized, only 7 acquired a strain different from the original one. Approximately one-third of the carriers eliminated the colonization, and a similar number became newly colonized. Some of these events probably represent detection failure rather than genuine colonization loss or acquisition. Lower bacterial counts were associated with increased probability of eliminating the colonization. We have confirmed a high mutation rate in the spa locus: 6 out of 53 strains underwent mutation in the spa locus. There was no overall change in S. aureus genotype composition.

Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhône Valley.

Staphylococcus aureus is a major bovine mastitis pathogen. Although the reported antimicrobial resistance was generally low, the emergence of new genetic clusters in bovine mastitis requires examination of the link between antimicrobial resistance and genotypes. Here, amplified fragment length polymorphism (AFLP) profiles and standard antimicrobial resistance profiles were determined in order to characterize a total of 343 S. aureus cow mastitis isolates from two geographically close regions of Switzerland and France. AFLP profiles revealed similar population compositions in the two regions, with 4 major clusters (C8, C20, C97, and C151), but the proportions of isolates in each cluster significantly diverged between the two countries (P = 9.2 × 10⁻⁹). Antimicrobial resistance was overall low (< 5% resistance to all therapeutically relevant molecules), with the exception of penicillin resistance, which was detected in 26% of the isolates. Penicillin resistance proportions differed between clusters, with only 1 to 2% of resistance associated with C20 and C151 and up to 70% associated with bovine C97. The prevalence of C20 and C8 was unexpectedly high and requires further investigation into the mechanism of adaptation to the bovine host. The strong association of penicillin resistance with few clusters highlights the fact that the knowledge of local epidemiology is essential for rational choices of antimicrobial treatment in the absence of susceptibility testing. Taken together, these observations argue in favor of more routine scrutiny of antimicrobial resistance and antibiotic-resistant clones in cattle and the farm environment.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans.

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Methicillin-susceptible Staphylococcus aureus endocarditis isolates are associated with clonal complex 30 genotype and a distinct repertoire of enterotoxins and adhesins.

BACKGROUND: Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. METHODS: IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. RESULTS: 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). CONCLUSIONS: MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study.

Improved multiple-locus variable-number tandem-repeat assay for Staphylococcus aureus genotyping, providing a highly informative technique together with strong phylogenetic value.

We describe an improved multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for genotyping Staphylococcus aureus. We compare its performance to those of multilocus sequence typing (MLST) and spa typing in a survey of 309 strains. This collection includes 87 epidemic methicillin-resistant S. aureus (MRSA) strains of the Harmony collection, 75 clinical strains representing the major MLST clonal complexes (CCs) (50 methicillin-sensitive S. aureus [MSSA] and 25 MRSA), 135 nasal carriage strains (133 MSSA and 2 MRSA), and 13 published S. aureus genome sequences. The results show excellent concordance between the techniques' results and demonstrate that the discriminatory power of MLVA is higher than those of both MLST and spa typing. Two hundred forty-two genotypes are discriminated with 14 VNTR loci (diversity index, 0.9965; 95% confidence interval, 0.9947 to 0.9984). Using a cutoff value of 45%, 21 clusters are observed, corresponding to the CCs previously defined by MLST. The variability of the different tandem repeats allows epidemiological studies, as well as follow-up of the evolution of CCs and the identification of potential ancestors. The 14 loci can conveniently be analyzed in two steps, based upon a first-line simplified assay comprising a subset of 10 loci (panel 1) and a second subset of 4 loci (panel 2) that provides higher resolution when needed. In conclusion, the MLVA scheme proposed here, in combination with available on-line genotyping databases (including http://mlva.u-psud.fr/), multiplexing, and automatic sizing, can provide a basis for almost-real-time large-scale population monitoring of S. aureus.

Sternal wound infection after heart operations in pediatric patients associated with nasal carriage of Staphylococcus aureus.

A cluster of six pediatric cases of deep-seated Staphylococcus aureus infection after heart operations prompted us to perform molecular typing of the S. aureus isolates by pulsed-field gel electrophoresis. This revealed the presence of genotypically distinct isolates in four of the six patients. Isolates of two patients were genotypically identical. All patients carried S. aureus in the anterior nares. In each patient, the banding pattern of deoxyribonucleic acid in these isolates was indistinguishable from that in strains isolated from blood or wound cultures. Molecular typing with pulsed-field gel electrophoresis ruled out nosocomial transmission of S. aureus between four patients; at the same time, it provided evidence for an association between nasal colonization and postoperative wound infection. Epidemiologic investigation of potential links between two patients with identical isolates did not provide any evidence for nosocomial transmission of S. aureus between these patients. Because nasal colonization with S. aureus may be a risk factor for surgical wound infection in pediatric patients undergoing heart operations, preoperative decolonization appears to be warranted.