Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis

A total of 71 Staphylococcus aureus strains isolated from bovine mammary glands were identified and subtyped. The methods used to differentiate between the S. aureus isolates were the DNA polymorphism pattern after amplification with a Polymerase Chain Reaction using several primer combinations and phage typing. The DNA fingerprinting technique using RAPD, ERIC1R and ERIC primers proved to be useful in differentiating isolates of S. aureus. Differentiation of isolates using phage typing gave no additional information compared to the DNA technique. The outbreak of S. aureus in the herd studied was mainly caused by one S. aureus strain. Other strains were only found on three occasions, twice in subclinical infections and once from a case of clinical mastitis. In the latter case the dominant strain was isolated from a different quarter of the same cow. Four of the ten cows studied suffered from clinical mastitis. From those four cows, three remained infected with the same S. aureus strain despite antibiotic treatment.     

Evaluation of pulsed-field gel electrophoresis as a typing system for Candida rugosa: comparison of karyotype and restriction fragment length polymorphisms

Nosocomial infections with Candida species have emerged as an increasingly important cause of morbidity and mortality in intensive care units. Ten Candida rugosa isolates from a previously documented cluster of C. rugosa infections in one hospital (nine burn unit isolates and one isolate from another hospital ward) and eight C. rugosa isolates recovered in a referral fungus testing laboratory (comparison isolates) from distinct geographic areas were investigated by molecular techniques. Isolates were from multiple anatomic sites. Pulsed-field gel electrophoresis (PFGE) of whole-cell DNA was performed with the 18 C. rugosa isolates as a marker of strain identity. The PFGE karyotypes of the C. rugosa isolates were demonstrated from four to seven chromosome bands. Karyotyping revealed the same PFGE pattern for the nine outbreak isolates from the burn unit, confirming clonal strain transmission. The isolate from the other hospital ward had a distinct karyotype. Distinct PFGE karyotype patterns were demonstrated for the eight comparison isolates. Restriction fragment length polymorphisms (RFLP) generated from whole-cell DNA digested with SfiI demonstrated the same RFLP pattern among outbreak isolates. Among comparison isolates, karyotyping distinguished some isolates that were indistinguishable by RFLP patterns. Karyotyping by PFGE appears to be the most useful molecular typing tool for discrimination among strains of C. rugosa and will be a useful marker for evaluating the epidemiology of future C. rugosa infections.     

Epidemiology of an unusually prolonged outbreak of typhoid fever in Terrassa, Spain

An unusually prolonged outbreak of typhoid fever, from 1988 to 1994, in Terrassa (Barcelona, Spain), was caused by a casual food handler who was a carrier. The pattern of this outbreak suggested intermittent low-level exposure to Salmonella typhi. We found 70 patients with S. typhi infections, 52 of whom were available for study. Medical records were reviewed and patients were interviewed with use of a standard questionnaire. Phage typing and pulsed-field gel electrophoresis (PFGE) for strain subtyping were used to confirm the epidemiological data. The 27 outbreak strains shared the same phage type and the same PFGE pattern. Four sporadic strains shared the same phage type as the outbreak strain. PFGE was found to be useful for differentiating strains for epidemiological purposes.     

Different distribution of H1-H2 Epstein-Barr virus variant in oropharyngeal virus and in biopsies of Hodgkin''s disease and in nasopharyngeal carcinoma from Algeria

Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993-1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections. The same DNA profiles within the isolates from the three episodes were obtained by both techniques. The Apal and Smal PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes. However, RAPD analysis with primer UBC-127 distinguished between these L. monocytogenes isolates.     

Typing, resistance behavior and occurrence of methicillin-resistant Staphylococcus aureus strains in a surgical intensive care unit

Over a period of three years, the frequency of the appearance of methicillin-resistant S. aureus strains (MRSA) was observed on a surgical intensive care unit. During this above-mentioned period of investigation it came to a heaped occurrence of nosocomial infections on this ICU with altogether 332 S. aureus-stems being isolated from different patient specimen. 204 (61.5%) of these were resistant against methicillin and could be divided into 48 first- and 156 follow-up-isolates. The thereupon accomplished differentiation of the 48 MRSA-first isolates by means of lysotyping and the pioneered GenePath Strain Typing System for a standardized pulsed-field-gel-electrophoresis (PFGE) gave the proof of 7 different MRSA-types. Around 7 different, in part parallel chains of infection on this ICU were observed, which could be led back to different strains. In reference to all analyzed S. aureus, an especially high rate (90%) of MRSA on this ICU could be isolated in taken wound-swabs, followed by 83.3% MRSA at catheter tips and 71,9% in tracheal and bronchial secretion. A consideration of the antibiotic susceptibility yielded, that also gentamicin and the quinolones showed an in-vitro resistance against MRSA, while fosfomycin, fusidic acid, chloramphenicol and trimethoprim/sulfamethoxazole reached positive responding rates between 80 and 100%. On the other hand, presently still 100% of the explored MRSA-strains are susceptible for glycopeptides such as vancomycin and teicoplanin. Because of intensive hospital hygienic measures the number of newly isolated MRSA could be reduced clearly on this ward.     

Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time

Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of chromosomal DNA was used to confirm the persistence of methicillin-sensitive Staphylococcus aureus isolates in the sputum of 25 cystic fibrosis patients in five French hospitals. Three-to-eight consecutive isolates, with the same esterase electrophoretic type isolated from each patient over a period of 12-28 months, were analysed. Consecutive isolates with indistinguishable PFGE profiles were found in 12 patients (48%) and consecutive isolates with similar PFGE profiles showing minor differences of one-to-four fragments (similarity coefficient >/=84%) were found in 11 patients. Consecutive isolates with different PFGE profiles were obtained from only two patients, but the profiles found in each patient were more closely related to each other than to other profiles. The results were in agreement with esterase electrophoretic typing for 23 patients, and we considered that those patients were infected with a single persistent strain. For any given patient, variations in antibiotypes and phage types of consecutive isolates were not associated with major genotypic variations. PFGE is useful in confirming the persistence of S. aureus strains in cystic fibrosis patients over long periods.     

Gaining control: family members relate to persons with severe mental illness

Nine isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected in a Warsaw hospital in 1996 were typed by phenotypic (resistograms) and genotypic (PFGE and plasmid restriction analysis-REAP) methods. Twenty-four (MRSA) strains collected in this hospital during a period of the same duration in 1992 and typed earlier using resistograms and PFGE were also typed by REAP. Comparison of typing results obtained for isolates from 1992 and 1996 showed that strains characterised by PFGE patterns of two distinct types described as specific of the two clonally related groups of Polish MRSA in a multicentre study in 1992 are continuously present in the hospital. However, MRSA strains representing PFGE patterns not observed before were also found within the collection from 1996. REAP typing has proved to have a discriminatory power similar to that of PFGE analysis. Nevertheless, due to the lack of plasmids or difficulties in plasmid DNA isolation in 3 out of 33 studied strains, the typability of REAP turned out to be lower than that of PFGE.     

Genotyping of clinical isolates of MRSA by PFGE and AP-PCR

Genotyping of 67 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) separated from patients in a hospital in Mizusawa City in 1994 and 1995 was studied by pulsed field gel electrophoresis (PFGE) and arbitrarily primed-polymerase chain reaction (AP-PCR). Two main genotypes were observed by PFGE, and more than 70% of the 67 MRSA isolates produced coagulase type II. One group diverged well and gained higher tolerance in 1994, but was not isolated in 1995. The other group was continually isolated during the two-year period and showed moderate tolerance in 1994, and higher tolerance in 1995. AP-PCR was able to classify the genotypes of MRSA into 6 subgenotypes under the present conditions, which supported the results obtained by PFGE. These results suggest that AP-PCR could become a convenient and useful typing method by improving both sequence and length of a primer.     

Insertion possibility of 16S-23S space amplification and random amplified polymorphic DNA analysis for typing of methicillin-resistant Staphylococcus aureus strains in the context of nosocomial infections

Within the scope of the present study n = 183 MRSA isolates from the extended area of Düsseldorf and n = 93 international MRSA strains from seven different countries were typed by pulsed-field gel electrophoresis and two PCR methods (RAPD and 16S-23S-spacer amplification). The isolates could be subdivided into 30 different types by PFGE, into 21 by means of RAPD and 18 by 16S-23S-spacer amplification. PFGE had the highest discriminatory potential, however, a combined use of the three typing methods allows a more detailed differentiation even of those isolates with identical PFGE pattern. Both amplification procedures were rapid, easy in handling with reproductable results. For a temporary epidemiological analysis within 24 hours, both amplification methods could be combined. In case the investigated isolates were still suspected of showing a "clonal identity", they should be analysed by additional PFGE (lasting about four days). Although the international isolates were chosen by random selection, several MRSA strains with identical pattern could be found in different countries of the world. Some RAPD-, spacer- and PFGE pattern were constant over many years. This reflects a high genetic stability of single strains.     

Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms

IS256 elements are present in multiple copies in the staphylococcal genome, either flanking the transposon Tn4001 or independent of it. PCR-based analysis of inter-IS256 spacer polymorphisms was developed for typing of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis strains. Using SmaI macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) as the reference method for MRSA typing, excellent reproducibility (100%), discriminatory power (97%), and in vivo stability were observed. Good concordance of the results with those of other molecular typing methods was found for two MRSA collections. Inter-IS256 PCR analysis of a U.S. collection of MRSA strains (n = 36), previously characterized by 15 typing methods, showed more limited discrimination. Agreement was 78% with PFGE analysis and 83% with ribotyping (HindIII). Analysis of a second set of Belgian MRSA strains (n = 17), categorized into two widespread epidemic clones by PFGE analysis, showed 65% agreement. For typing of S. epidermidis strains (n = 26), inter-IS256 PCR showed complete typeability (100%) and good discriminatory power (85%). Inter-IS256 PCR analysis is proposed as an efficient molecular typing assay for epidemiological studies of MRSA or S. epidermidis isolates.     

Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.     

Impact of control measures on the course of an outbreak of methicillin-resistant Staphylococcus aureus

BACKGROUND: Outbreaks of nosocomial infection by methicillin resistent Staphylococcus aureus (MRSA) are a problem in many hospitals with the control measures to be adopted being controversial. An outbreak of MRSA in a 550-bed university hospital is herein described and the impact of the adopted control measures on the evolution of the epidemic in the general hospitalization area (GHA) was analyzed. PATIENTS AND METHODS: The adopted control measures in the GHA were: microbiologic surveillance, cutaneous isolation measures, treatment of nasal carrier, and the early discharge of the cases. Hand washing was reinforced and a study of carriers was carried out on detection of sporadic cases (not related to the ICU). A molecular study of 70 strains of MRSA was performed with analysis of total plasmids, plasmid restriction pattern and chromosomic DNA analysis by pulsed field gel electrophoresis (PFGE). RESULTS: From December 1990 to December 1993, 273 cases of MRSA were reported. One hundred seventy-two cases originated in the ICU and 101 cases in the GHA (sporadic cases). The incidence of MRSA in 1991-1993 was 13.6, 14.3, and 6.6% in the ICU and 0.17, 0.36, and 0.15% in the GHA, respectively. Molecular study of MRSA isolates (1991 and 1992) demonstrated two plasmid and two chromosomic patterns. The latter had a similarity coefficient > 0.90, probably belonging to the same "clone". CONCLUSIONS: Despite the control measures adopted in the GHA the outbreak of MRSA originated in the ICU thereafter extending to the GHA. The rates of colonization detected, however, remained stable during the 3 years studied. On the other hand, the observation of a single "clone", responsible for the epidemic, suggest that most of the sporadic cases were autoctonous and due to failure in fulfillment of the established norms.     

MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression

A. baumannii is a multiresistant bacteria which is recognised as responsible for nosocomial infections and hospital outbreaks. The control of these outbreaks depends on the strain's typing and on the fight's policy against nosocomial infections. An outbreak of A. baumannii is occurred to patients who were hospitalized in Centre Hospitalier de Versailles. To investigate this outbreak, we have determined the biotype (Bouvet's method), the succeptibility pattern (disk diffusion and agar dilution results were analysed with the hierarchical classification and main component analysis) and the total DNA macrorestriction pattern (Pulse Field Gel Electrophoresis using SmaI restriction enzyme). A risk factors for A. baumannii acquisition were delineated in case-control study. During 2 years, 38 patients have been infected or colonized to A. baumannii. Thirty two patients were hospitalized in ICU. We studied 38 non repetitive clinical isolates and 9 strains of the patient's rooms. Four biotypes were defined by the Bouvet's typing method. Fourteen groups were obtained when succeptibility results were analysed with the hierarchical classification and 6 with the main composant analysis. The molecular typing permit us to define 4 epidemic and 6 sporadic strains. All the epidemic strains were isolated on ICU hospitalized patients. Our study has shown wide contamination in patient's rooms (Water tap, dry surfaces, patient's mattresses...). Environmental objects have been a major risk factor for A. baumannii acquisition. The control of this outbreak has been possible by application of hygienic measures (hands washing, isolment, meticulous cleaning of the ICU and environmental controls). No new case is occurred in the last year. Typing methods and case-control study are necessary to investigate cross-infections and take efficient measures against these outbreaks.     

Induction and measurement of antisperm antibody responses in vitro

We did a statistical study of 294 strains of Staphylococcus aureus (S. aureus) isolated from skin infections during the period from January of 1989 to December of 1991 in the Department of Dermatology, Kansai Medical University Hospital. We especially examined methicillin-resistant S. aureus (MRSA) from the point of view of incidence, variety of skin infections with MRSA, coagulase type, phase type, and resistance against antimicrobial agents. The frequency of isolation of MRSA has been increasing. In 1991, the proportion of MRSA isolates among all S. aureus strains isolated from skin infections was 41.5%. MRSA was isolated most often from infectious decubitus. Coagulase type II and phage group NT (not typable) MRSA were most frequently isolated. The resistance of MRSA to OFLX and IMP/CS had remarkably increased. Notably, the resistance to MINO was low before 1991.     

Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.     

Genetic diversification of methicillin-resistant Staphylococcus aureus as a function of prolonged geographic dissemination and as measured by binary typing and other genotyping methods

The aim of the present study was to determine the extent of genome evolution among methicillin-resistant Staghylococcus aureus (MRSA) strains. Three different collections of strains were analysed, comprising locally, nationally and internationally disseminated genotypes. Various genotyping assays displaying different levels of resolution were used. Geographically and temporally diverse MRSA strains comprised the international group. MRSA strains recovered during an outbreak in a New York City hospital and Portuguese MRSA isolates, all resembling the so-called Iberian clone, were included in the local and national collections, respectively. Genotypes were determined by genome scanning typing techniques and procedures which analyse specific DNA elements only. The outbreak strains showed subclonal variation, whereas the Portuguese isolates displayed an increased number of genotypes. Among the epidemiologically unrelated MRSA strains, the different genotyping techniques revealed a wide heterogeneity of types. Different typing techniques appeared to show different levels of resolution, which could be correlated with the extent of geographic spread; the more pronounced the spread, the higher the degree of genome evolution. Binary typing and randomly amplified polymorphic DNA analysis are the typing methods of choice for determining (non)identity among strains that have a recent common ancestor and have undergone yet limited dissemination.     

Molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy was studied in a five-month period in 1996, during which all S. aureus isolated were collected. All MRSA isolates (95) and a sample of methicillin-susceptible S. aureus (20) were typed with a variety of phenotypic and genotypic methods. Clonal identities were determined by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests and, for MRSA isolates, by probing ClaI digests with a mecA probe and a Tn554 probe. Overall, MRSA represented 32.3% of all isolates, with very high percentages from the intensive care units (adult and neonatal). PFGE after restriction with SmaI resolved genomic DNA of 95 MRSA strains into 26 major PFGE patterns. The use of southern blot hybridization of ClaI genomic digests with mecA and Tn554 allowed us a significant increase in discrimination, differentiating at least 32 different clones. Two major clones, however, each sharing common ClaI-mecA and Tn554 type and PFGE pattern as well as a common resistance phenotype, represented more than 50% of all MRSA isolates. The recovery of these two clones in the majority of the isolates of adult and neonatal intensive care units, respectively, is indicative of typical nosocomial outbreaks and clonal spread. It is concluded that intensive care units are major areas requiring preventative interventions.     

Recent data on molecular genetics of neuroendocrine tumors

Between December 1994 and April 1995, a nosocomial outbreak caused by a multi-resistant Acinetobacter baumannii, occurred on a surgical ward in our hospital. The organism was isolated from 13 patients, eight of whom were infected whereas the others were colonized. Twelve isolates were compared by cell envelope protein electrophoretic profiles and AFLP, a recently described DNA fingerprinting method. Both methods indicated that this outbreak was caused by spread of a single strain, which was identified as A. baumannii by amplified ribosomal DNA fingerprinting (ARDRA). A case-control comparison was performed to identify risk factors associated with nosocomial acquisition of A. baumannii. Risk factors for cross-colonization were length of stay, surgery, wounds and treatment with broad-spectrum antibiotics. Cross-infection with A. baumannii among patients occurred despite implementation of stringent infection control measures. The outbreak was controlled after temporary closure of the surgical ward for disinfection purposes. Patients admitted on a general surgical ward colonized or infected with multi-resistant A. baumannii strains should alert the hospital infection control team, and prompt implementation of strict infection prevention measures to prevent further spread is advised.     

Molecular markers for differentiation of multiresistant Klebsiella pneumoniae isolates in a pediatric hospital

OBJECTIVE: To study the spread of extended-spectrum beta-lactamase-producing, but aminoglycoside-susceptible, Klebsiella pneumoniae strains in our hospital over an 8-month period, by using two genotypic markers. DESIGN: Ribotyping (using two endonucleases) and randomly amplified polymorphic DNA analysis (RAPD; using two different 10-mer primers) were applied to the epidemiological typing of clinical K pneumoniae isolates from stools, ileal fluid, or urine of hospitalized children. SETTING AND PATIENTS: The surgical intensive-care ward (S1: 9 patients, 17 isolates), surgical unit (S2: 2 patients, 2 isolates), and gastroenterology ward (GE: 1 patient, 1 isolate) of the Robert Debré Hospital of Paris, France. RESULTS: Ribotyping of the 20 clinical isolates, the type strain of the species, and two epidemiologically unrelated isolates with EcoRI and HindIII revealed 6 and 5 different patterns, respectively. Six ribotypes were identified by using these two enzymes. RAPD generated 6 distinct patterns, in complete agreement with ribotyping. Our genotypic results showed that 11 patients from wards S1, S2, and GE harbored genotypically related strains, suggesting nosocomial transmission and cross-colonization between and within the three wards. CONCLUSIONS: Ribotyping and RAPD appear to be reliable methods for distinguishing K pneumoniae strains. The spread of one strain of K pneumoniae in different units of our hospital was demonstrated by both methods. However, RAPD has the advantage of simplicity and rapidity conferred by polymerase chain reaction.     

Tracing the spread of methicillin-resistant Staphylococcus aureus by Southern blot hybridization using gene-specific probes of mec and Tn554

In a community hospital in Brooklyn, New York, over a 3-year period, 79 methicillin-resistant Staphylococcus aureus (MRSA) isolates from five different case clusters were subtyped by Southern blot hybridization with two previously characterized gene probes, mec and Tn554. Together, the genotyping enabled the hospital infection control team to differentiate simultaneous MRSA clusters in the surgical intensive care unit (type I:A) and the open heart unit (type II:J), document the spread of one strain (type I:A) between roommates, identify an endemic strain (type II:J) from cardiac monitors and medical personnel, and identify an unrelated outbreak strain (type II:NH) in the labor and delivery unit. On the basis of this investigation it is clear that the routine DNA fingerprinting of MRSA in health care facilities, to monitor their spread and identify cases of nosocomial infections, is an important infection control measure.