Transient and persistent increases in protein phosphatase activity during long-term depression in the adult hippocampus in vivo

In a 34-month prospective study to determine the prevalence of Staphylococcus aureus small colony variants (SCVs) in cystic fibrosis (CF) patients, S. aureus SCVs or SCVs plus normal S. aureus were recovered from 26 of 78 patients; 27 patients harbored only normal S. aureus. By pulsed-field gel electrophoresis, clonal identity was demonstrated of SCV and normal strains isolated at the same time and of multiple S. aureus SCV and normal strains in consecutive specimens from individual patients. All S. aureus SCVs were resistant to antifolate antibiotics, while the corresponding parent strains were susceptible, and in 11 of 12 SCV/normal pairs, gentamicin was less active against S. aureus with the SCV phenotype than against the normal isolate. Analysis of the underlying auxotrophism of SCVs revealed hemin, thymidine, and/or menadione dependencies. Thus, S. aureus SCVs are highly prevalent in respiratory secretions of CF patients, persist over extended periods, and may contribute to S. aureus persistence in CF patients.     

Variation in metabolic enzyme activity of persistent Haemophilus influenzae in respiratory tracts of patients with cystic fibrosis

Haemophilus influenzae organisms were isolated from sputum specimens prospectively collected from 40 patients with cystic fibrosis during 2 years to study variations in the metabolic enzyme activities of persistent H. influenzae strains as determined by biotyping. In total, 97 distinct H. influenzae strains without variations in their major outer membrane protein (MOMP) patterns and 73 MOMP variants derived from 30 of these distinct strains were obtained. Twelve distinct strains and 42 MOMP variant strains were isolated at multiple time points during the study period, indicating the persistence of these strains. Among the 54 persistent H. influenzae strains, 22 (41%) strains with stable MOMP compositions showed random variations in biotypes. In 39 of 103 (38%) H. influenzae strains, biotype changes coincided with MOMP variations. Biotype variations were the result of both the loss and the acquisition of enzyme activities. The results of the study indicate that changes in metabolic enzyme activity occur randomly during the persistence of H. influenzae organisms in cystic fibrosis patients, irrespective of MOMP variations.     

Streptococcus bovis clone causing two episodes of endocarditis 8 years apart

A patient had endocarditis caused by Streptococcus bovis twice 8 years apart. According to pulsed-field gel electrophoresis (PFGE) the two isolates were identical. Seven unrelated blood isolates of S. bovis yielded unique PFGE patterns. Considering this heterogeneous population structure our findings demonstrate the long-term persistence of an S. bovis clone in a patient with recurrent endocarditis.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping

Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented. Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients. This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States. Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis. Frequently RTs occurred in more than a single ET. Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs. Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources. This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P. cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices. Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.     

Molecular analysis by pulsed-field gel electrophoresis and antibiogram of Streptococcus pneumoniae serotype 6B isolates from selected areas within the United States

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (+/-1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.     

Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis

We examined genetic variation among 78 clinical isolates of Bordetella pertussis, including 54 strains recovered during a 1986 pertussis epidemic. A total of 16 pulsed-field gel electrophoresis (PFGE) profiles, generated with each of three different enzymes (XbaI, SpeI, and DraI), were obtained from the epidemic and sporadic isolates included in the study. Indistinguishable profiles were seen among strains unrelated temporally or geographically, as well as among strains isolated sporadically from the same geographic areas. All isolates from the epidemic had indistinguishable PFGE profiles. The PFGE pattern of the epidemic strains was shared with only 1 of 25 strains isolated independently of the outbreak. This isolate was cultured from a specimen from a laboratory scientist who had been working with the epidemic strains, further implicating the usefulness of PFGE for the epidemiologic study of clinical strains of B. pertussis. Differences in PFGE profiles for single epidemic strains occurred occasionally upon repeated passage on agar medium, suggesting that subculturing of initial isolates should be minimized before pulsed-field analysis.     

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.     

Molecular analysis of multiple isolates of the major serotypes of group B streptococci

Serotyping of clinical isolates is a widely used technique for epidemiologic study of group B streptococcal infections. However, serotyping cannot definitively determine epidemiologically related or unrelated isolates. We investigated the use of restriction endonuclease analysis (REA) with both conventional agarose gel electrophoresis (AGE) and pulsed-field gel electrophoresis (PFGE) in 50 isolates of the major serotypes of group B streptococci. Single digestion with HindIII and HaeIII and double digestion with HindIII and then EcoRI were used for conventional AGE, and digestion with SmaI was used for PFGE. The molecular profile of one strain was compared with those of the strains within the same serotype as well as with the profiles from strains of different serotypes. Among 10 type Ia, Ia/alpha, Ia/alpha+beta, and Ia/R1 isolates and depending on the restriction enzyme used, we found between five and six REA patterns by conventional AGE and seven by PFGE; among 4 type Ib/alpha+beta isolates we found 2 to 4 REA patterns by conventional AGE and 4 by PFGE; among 21 type II, II/alpha, II/beta, II/alpha+beta, and II/R4 isolates, we found 11 REA patterns by both AGE and PFGE; and among 14 type III, III/R1, and III/R4 isolates, we found from 7 to 12 different REA patterns by AGE and 10 by PFGE. In total, among 13 serotypes and one nontypeable strain, we found 29 to 31 REA patterns by conventional AGE and 33 by PFGE. A particular REA pattern within a serotype was different from the patterns found in the other serotypes, suggesting that REA analysis by using conventional AGE or PFGE is a sensitive method for analyzing genetic relatedness and diversity in group B streptococci and has potential value in molecular epidemiologic studies.     

Characterization of IS1245 for strain typing of Mycobacterium avium

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had > or = 10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.     

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan

Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum beta-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.     

Nasal carriage of staphylococcus aureus and epidemiology of surgical-site infections in a Sudanese university hospital

Surgical site infections (SSI) due to Staphylococcus aureus among 256 male and 158 female patients (mean age, 28 years) undergoing elective surgery at the Soba University Hospital (Khartoum, Sudan) were studied. During an 11-month study period all patients were analyzed for nasal carriage of S. aureus at the time of admission. Follow-up of the development of SSI proceeded until 4 weeks after the operations. In addition, nasal swabs were obtained periodically during the same period from 82 members of the staff. In order to discriminate autoinfection from cross infection, bacterial isolates were typed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments, and restriction fragment length polymorphism analysis of the protein A and coagulase genes. Preoperative cultures revealed the presence of S. aureus in the noses of 98 patients (24%). The overall number of postsurgical wound infections in the entire group was 57 (14%), 24 of which were due to S. aureus. Only 6 of the 98 nasal S. aureus carriers suffered from wound infections by the same species. In these six cases the infecting strain could not be genetically discriminated from the nasal inhabitant, substantiating autoinfection. However, nasal carriage of S. aureus is not a significant risk factor for the development of SSI in this setting (6 of 98 patients with autoinfection versus 18 of 316 patients [414 - 98 patients] with cross infection; P = 0.81), most probably due to the fact that noncarriers are at a significant and relatively large risk for acquiring an independent S. aureus SSI. The other S. aureus strains causing SSI showed a high degree of genetic heterogeneity, demonstrating that it is not an epidemic strain that is causing the SSI. Among the staff personnel screened, 47.4% did not carry S. aureus in the nose at any time during the study period, whereas 13. 2% persistently carried a single strain in the nose. Another 39.5% could be classified as intermittent carriers. When strains derived from staff personnel were genetically typed, it was demonstrated that most of the strains represented genetic variants clearly differing from the isolates causing SSI. On the other hand, possible cross colonization among staff personnel and even cross infection from staff personnel to patients or from patient to patient were demonstrated in some cases, but epidemic spread of a single strain or a few clonally related strains of S. aureus could be excluded.     

Stability of strain genotypes of Burkholderia pseudomallei from patients with single and recurrent episodes of melioidosis

The constancy of strain genotypes of multiple isolates of Burkholderia pseudomallei from 13 patients with melioidosis was examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of DNA. Seven of 8 patients with single episodes of melioidosis each yielded genetically identical isolates and only one of five patients with recurrent episodes was infected with a new strain clearly distinct from the original primary strain. Variation was observed in PFGE patterns of primary and relapse isolates of another patient but this was insufficient to define genetically distinct strains. We conclude that most patients with single or multiple episodes of melioidosis retain a single strain.     

Comparison of traditional and molecular methods for typing nontoxigenic strains of Corynebacterium diphtheriae

Thirty-eight nontoxigenic strains of Corynebacterium diphtheriae isolated between 1987 and 1992 from clinical specimens of French patients were typed by biotyping, antibiograms, bacteriophage typing, ribotyping, and restriction analysis by pulsed-field gel electrophoresis (PFGE). Excellent correlation occurred between the genotypes defined by PFGE SfiI profiles or by ribotype BstEII profiles. Genotyping revealed seven genotype patterns among the 26 biotype mitis isolates, five among the nine biotype gravis isolates, and three among the three biotype belfanti isolates. Phage typing was nonreactive for nine of the 38 isolates. A combination of all the typing methods led to the identification of 19 different types of Corynebacterium diphtheriae.     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

Fluorescent amplified-fragment length polymorphism analysis of an outbreak of group A streptococcal invasive disease

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was carried out for an outbreak of group A streptococcal (GAS) invasive disease. Streptococcal genomic DNAs were digested with endonucleases EcoRI and MseI, site-specific adaptors were ligated, and PCR amplification was carried out with an EcoRI adaptor-specific primer labelled with fluorescent dye. Amplified fragments of up to 600 bp in size were separated on a polyacrylamide sequencing gel which contained internal size markers in each lane. These data were automatically scanned and analyzed, fragments were precisely sized (+/-1 bp), and electropherograms were generated for each genome with GeneScan 2.1 software. All isolates were compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP profiles, compared with 12 macrorestriction profiles by pulsed-field gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of invasive disease in a care-of-the-elderly system. It provided an automated analysis of the whole genome of bacterial isolates. It was reproducible, more discriminatory, and capable of higher throughput than other molecular typing methods. Given agreed conditions, FAFLP would be reproducible between laboratories for rapid characterization of outbreak strains.     

Acute opioid dependence in the cardiovascular system of the spinal rat

A cluster of infections caused by Enterobacter cloacae was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of similar antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of a single strain existed. All 12 E. cloacae isolates from the NICU and 50 nonrelated strains were fingerprinted by small-fragment restriction endonuclease analysis (SF-REA) of EcoRI DNA digests. Selected isolates were further characterized by pulsed-field gel electrophoresis (PFGE) of NotI- or XbaI-generated genomic restriction fragments. Epidemiologically unrelated strains were clearly discriminated by both methods. Results achieved by SF-REA and PFGE revealed that of the 12 isolates from the NICU, 11 belonged to the same genotypic cluster. Since all reagents and equipment for both techniques are commercially available, DNA fingerprinting by SF-REA or PFGE is proposed as a useful tool in the microbiology laboratory for investigating the epidemiological relatedness of E. cloacae strains of clinical and environmental origin.     

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.     

Comparison of genomic methods for differentiating strains of Enterococcus faecium: assessment using clinical epidemiologic data

Genomic DNA extracted from 45 vancomycin-resistant Enterococcus faecium (VRE) isolates was cleaved with HindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single "ideal" method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection.     

In vitro activities of meropenem, PD 127391, PD 131628, ceftazidime, chloramphenicol, co-trimoxazole, and ciprofloxacin against Pseudomonas cepacia

In a study of 110 Pseudomonas cepacia isolates from patients without cystic fibrosis, the in vitro potencies of three new compounds, meropenem, PD 127391, and PD 131628, were comparable to those of ceftazidime and ciprofloxacin and exceeded those of chloramphenicol and co-trimoxazole. The MICs of ceftazidime, ciprofloxacin, meropenem, and the PD compounds for 90% of strains tested were < or = 4 micrograms/ml, whereas they were 32 micrograms/ml for chloramphenicol and co-trimoxazole. Data for 20 isolates from patients with cystic fibrosis indicated that the isolates were less susceptible to all seven antibiotics tested, with the most active compounds being meropenem and PD 127391.