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.

Molecular typing of mycobacteria isolated from extrapulmonary tuberculosis patients at Debre Birhan Referral Hospital, central Ethiopia.

Abstract Background: Extrapulmonary tuberculosis (EPTB) constitutes about 10% to 20% of all cases of tuberculosis in immunocompetent patients and more than 50% of the cases in HIV-positive individuals worldwide. Little information is available on the clonal diversity of Mycobacterium species in Ethiopia from EPTB. Methods: This study was carried out on smear-negative EPTB patients to molecularly characterize Mycobacterium tuberculosis complex strains. A questionnaire, smear staining, culture, deletion typing, and spoligotyping were employed. Results: The proportional distribution of EPTB and isolates did not vary substantially (p > 0.05) amongst the socio-demographic parameters considered in the current investigation. Out of 98 fine needle aspirates processed for culture, 36.7% (36/98) were positive for mycobacterial growth. Further speciation of those culture-positive isolates showed that 88.9% were M. tuberculosis and the remaining could be non-tuberculous mycobacterial species. Spoligotyping revealed 16 clusters out of which 2 were new to the SITVIT database. The most dominant spoligotypes were SIT54, SIT53, and SIT149 in decreasing order. SIT54, SIT134, SIT173, SIT345, SIT357, SIT926, SIT91088, and SIT1580 were reported for the first time in Ethiopia. The family with the highest frequency identified was M. tuberculosis family T1, followed by family 33. Most of the strains belonged to Euro-American (61.4%) and Indo-Oceanic (36.3%) lineages. Conclusions: The present study shows the importance of M. tuberculosis as a major cause of EPTB in the study area. Moreover, the majority of isolates of M. tuberculosis were found in clusters, suggesting the possibility of the existence of recent transmission. This warrants strengthening of the control programs for EPTB in the study area.

Molecular epidemiology of predominant clones and sporadic strains of methicillin resistant Staphylococcus aureus in Switzerland and comparison with European epidemic clones.

OBJECTIVE: To assess the molecular epidemiology and risk factors of predominant clones and sporadic strains of methicillin-resistant Staphylococcus aureus (MRSA) in Swiss hospitals and to compare them with European strains of epidemic clones. MATERIAL AND METHODS: One-year national survey of MRSA cases. Analysis of epidemiological and molecular typing data (PFGE) of MRSA strains. RESULTS: In 1997, 385 cases of MRSA were recorded in the five Swiss university hospitals and in 47 community hospitals. Half of the cases were found in Geneva hospitals where MRSA was already known to be endemic. Molecular typing of 288 isolates (one per case) showed that 186 (65%) belong to four predominant clones, three of which were mostly present in Geneva hospitals. In contrast, the fourth clone (85 cases) was found in 23 hospitals (in one to 16 cases per hospital). The remaining 35% of the strains were clustered into 62 pulsed field gel electrophoresis types. They accounted for one to five patients per hospital and were defined as sporadic. Multivariate analysis revealed no independent risk factors for harboring a predominant versus a sporadic strain, except that transfer from a foreign hospital increases the risk of harboring a sporadic strain (OR, 42; 95% CI, 5-360). CONCLUSION: While cases with predominant clones were due to the local spread of these clones, most sporadic cases appear to be due to the continuous introduction of new strains into the country. With the exception of a transfer from a hospital outside Switzerland, no difference in the clinical or epidemiological characteristics was observed between patients harboring a predominant clone and those with a sporadic strain.

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations.

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize "high-risk" epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks.

Unusual spread of a penicillin-susceptible methicillin-resistant Staphylococcus aureus clone in a geographic area of low incidence.

We describe the unusual spread of a penicillin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) clone in hospitals in western Switzerland, where the incidence of MRSA is usually low. During a 2-year period, this clone had been responsible for several outbreaks and had been isolated from >156 persons in 21 institutions. Molecular typing by pulsed-field gel electrophoresis (PFGE) demonstrated that all of these isolates belonged to the same clone. In 1 of the outbreaks, involving 30 cases, the clone was responsible for at least 17 secondary cases. In contrast, during the period of the latter outbreak, 9 other patients harboring different MRSA strains, as assessed by PFGE, were hospitalized in the same wards, but no secondary cases occurred. These observations suggest that this clone, compared with other MRSA strains, had some intrinsic factor(s) that contributed to its ability to disseminate and could thus be considered epidemic.

Risk for Pneumocystis carinii transmission among patients with pneumonia: a molecular epidemiology study.

We report a molecular typing and epidemiologic analysis of Pneumocystis carinii pneumonia (PCP) cases diagnosed in our geographic area from 1990 to 2000. Our analysis suggests that transmission from patients with active PCP to susceptible persons caused only a few, if any, PCP cases in our setting.

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.

Molecular epidemiology of pseudomonas aeruginosae in intensive care units (ICUs) over a 10-year period (1998-2007)

Pseudomonas aeruginosa, une bactérie environnementale ubiquitaire, est un des pathogènes nosocomiaux les plus fréquents aux soins intensifs. La source de ce microorganisme peut être soit endogène, 2,6 à 24 % des patients hospitalisés étant colonisés au niveau digestif, soit exogène. La proportion des cas d'infections à P. aeruginosa d'origine exogène, donc secondaires à une transmission par manuportage ou par l'eau du réseau utilisée pour la toilette ou d'autres soins, reste débattue. Or une meilleure évaluation du taux d'infections exogènes est importante pour la mise en place de mesures de contrôle appropriées. Le but de cette étude était de déterminer sur une période de 10 ans les rôles respectifs des sources exogènes (robinets, autres patients) et endogène dans la colonisation et/ou l'infection par P.aeruginosa chez les patients des Soins Intensifs, ainsi que de documenter les variations épidémiologiques au cours du temps. L'étude a été menée dans les unités de Soins Intensifs du Centre Hospitalier Universitaire Vaudois (CHUV). Les patients colonisés et/ou infectés par P. aeruginosa entre 1998 et 2007ont été identifiés via la base de données du laboratoire de microbiologie. Ils ont été inclus dans l'étude s'ils étaient hospitalisés dans une des unités de Soins Intensifs, Durant cette période, des prélèvements pour recherche de P. aeruginosa ont été effectués sur des robinets des soins intensifs. Un typage moléculaire a été effectué sur toutes les souches cliniques et environnementales isolées en 1998, 2000, 2003, 2004 et 2007. Les patients inclus dans l'étude ont été répartis en quatre catégories (A-D) selon le résultat du typage moléculaire leur souche de P. aeruginosa. La catégorie A inclut les cas pour lesquels le génotype de P. aeruginosa est identique à un des génotypes retrouvé dans l'environnement. La catégorie B comprend les cas pour lesquels le génotype est identique à celui d'au moins un autre patient. La catégorie C comprend les cas avec un génotype unique et la catégorie D comprend les cas pour lesquels la souche était non disponible pour le typage. Les cas des catégories A et B sont considérés comme ayant une origine exogène. Au cours des années de l'étude, le nombre d'admissions aux soins intensifs est resté stable. En moyenne, 86 patients par année ont été identifiés colonisés ou infectés par P. aeruginosa aux Soins Intensifs. Durant la première année d'investigation, un grand nombre de patients colonisés par une souche de P. aeruginosa identique à une de celles retrouvées dans l'environnement a été mis en évidence. Par la suite, possiblement suite à l'augmentation de la température du réseau d'eau chaude, le nombre de cas dans la catégorie A a diminué. Dans la catégorie B, le nombre de cas varie de 1,9 à 20 cas/1000 admissions selon les années. Ce nombre est supérieur à 10 cas/1000 admissions en 1998, 2003 et 2007 et correspond à des situations épidémiques transitoires. Tout au long des 10 ans de l'étude, le nombre de cas dans la catégorie C (source endogène) est demeuré stable et indépendant des variations du nombre de cas dans les catégories A et B. En conclusion, la contribution relative des réservoirs endogène et exogène dans la colonisation et/ou l'infection des patients de soins Intensifs varie au cours du temps. Les facteurs principaux qui contribuent à de telles variations sont probablement le degré de contamination de l'environnement, la compliance des soignants aux mesures de contrôle des infections et la génétique du pathogène lui-même. Etant donné que ce germe est ubiquitaire dans l'environnement aqueux et colonise jusqu'à 15% des patients hospitalisés, la disparition de son réservoir endogène semble difficile. Cependant, cette étude démontre que son contrôle est possible dans l'environnement, notamment dans les robinets en augmentant la température de l'eau. De plus, si une souche multi-résistante est retrouvée de manière répétée dans l'environnement, des efforts doivent être mis en place pour éliminer cette souche. Des efforts doivent être également entrepris afin de limiter la transmission entre les patients, qui est une cause importante et récurrente de contamination exogène. - Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeru¬ginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in cat¬egories A and Β were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category Β varied from 1.9 to 20 cases per 1000 admissions. This num¬ber exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases con¬sidered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.

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.

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Comprehensive molecular profiling of lung adenocarcinoma.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method.

Although the molecular typing of Pseudomonas aeruginosa is important to understand the local epidemiology of this opportunistic pathogen, it remains challenging. Our aim was to develop a simple typing method based on the sequencing of two highly variable loci. Single-strand sequencing of three highly variable loci (ms172, ms217, and oprD) was performed on a collection of 282 isolates recovered between 1994 and 2007 (from patients and the environment). As expected, the resolution of each locus alone [number of types (NT) = 35-64; index of discrimination (ID) = 0.816-0.964] was lower than the combination of two loci (NT = 78-97; ID = 0.966-0.971). As each pairwise combination of loci gave similar results, we selected the most robust combination with ms172 [reverse; R] and ms217 [R] to constitute the double-locus sequence typing (DLST) scheme for P. aeruginosa. This combination gave: (i) a complete genotype for 276/282 isolates (typability of 98%), (ii) 86 different types, and (iii) an ID of 0.968. Analysis of multiple isolates from the same patients or taps showed that DLST genotypes are generally stable over a period of several months. The high typability, discriminatory power, and ease of use of the proposed DLST scheme makes it a method of choice for local epidemiological analyses of P. aeruginosa. Moreover, the possibility to give unambiguous definition of types allowed to develop an Internet database ( http://www.dlst.org ) accessible by all.

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.

Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007).

Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeruginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in categories A and B were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category B varied from 1.9 to 20 cases per 1000 admissions. This number exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases considered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.