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Use of molecular typing to study the epidemiology of Serratia marcescens.

Although Serratia marcescens is a well-known nosocomial pathogen, investigation of its hospital ecology has been limited by the lack of available typing techniques. During an investigation of the occurrence of this organism in a neonatal intensive care unit, we evaluated a number of such techniques. Using a selective medium, we conducted prospective surveillance of neonatal rectal colonization and environmental contamination with S. marcescens. In 8 months of surveillance, 5.1% (20 of 394) of the infants admitted to the unit became colonized. Most sink surfaces and drains were also culture positive. Differences between isolates could not be detected in biotypes from a commercial identification system (MicroScan) or by antibiograms, total protein fingerprints, or plasmid profiles. Serogrouping and genomic DNA restriction endonuclease analysis revealed the presence of six strains that colonized infants and a similar number of environmental strains. These two methods were concordant, with the exception that genomic DNA analysis demonstrated lack of relatedness between some strains within the same serogroup. DNA restriction endonuclease analysis was practical and reliable. The differences this method detected between environmental and neonatal strains provided strong evidence that the environment was not an important reservoir for S. marcescens in our neonatal intensive care unit.

Cross Infection↗

Epidemiological markers for epidemic strain and carrier isolates in an outbreak of nosocomial oxacillin-resistant Staphylococcus aureus.

An outbreak of nosocomial infections occurring in a postoperative intensive care unit was caused by a single strain of oxacillin-resistant Staphylococcus aureus. Six patients were infected, or colonized, by this strain, which was traced by using the following four epidemiological markers: antibiogram, bacteriophage type, capsular polysaccharide type, and esterase electrophoretic type. This strain was compared with S. aureus isolates obtained from the noses of 13 carriers from a group of 42 staff members. A good correlation in terms of phenotypic markers was found between the epidemic strain and a strain isolated from one carrier. Both exhibited the same pattern of multiple resistance as well as the same phage type, 77, capsular polysaccharide type, 5, and esterase electrophoretic type, 6. In contrast, an oxacillin-resistant strain, isolated from another carrier, differed from the epidemic strain by susceptibility to rifampin and by susceptibility to four additional bacteriophages. The other 11 strains isolated from carriers were susceptible to oxacillin and exhibited widely different phenotypes. These results confirm the interest of using several epidemiological markers to trace the spread of epidemic S. aureus strains and to delineate the carrier strains.

Bacterial Typing Techniques↗

Characterization of epidemic and nonepidemic Neisseria meningitidis serogroup A strains from Sudan and Sweden.

A random selection of 25 strains isolated during an epidemic caused by serogroup A Neisseria meningitidis in Sudan (1988), 3 preepidemic meningococcal strains (1985), and 26 serogroup A strains isolated from sporadic cases of meningitis in Sweden (1973 to 1987) were assessed for multilocus enzyme genotypes (ETs), DNA restriction enzyme patterns, outer membrane proteins, lipopolysaccharides, pilus formation, and antibiograms. All of the 25 Sudanese epidemic isolates and 22 of the Swedish strains were of the same or closely related ETs (ETs 3, 4, and 5), corresponding to clone III-1, which has been responsible for two pandemic waves in the last three decades. The earlier pandemic involved Scandinavia, and the last one caused an outbreak during the pilgrimage to Mecca, Saudi Arabia (August 1987), spreading to Sudan, Chad, and Ethiopia. The three Sudanese preepidemic isolates (1985) were clone IV-1 (sulfonamide susceptible), which has been resident in the African meningitis belt for the last 25 years. The uniformity of clone III-1 strains (all sulfonamide resistant) from Sudan and Sweden was confirmed by DNA restriction enzyme patterns. ETs 3, 4, and 5 from Sudan and Sweden had 86 to 100% similarity to a Swedish clone III-1 reference strain, whereas ETs 1, 2, 6, and 7 showed 50 to 80% similarity. Class 1 protein for clone III-1 showed serosubtype antigens P1.9 and P1.x, whereas ET6 strains (clone IV-1) had serosubtype P1.7. Lipopolysaccharides were variable in the Sudanese and Swedish strains. Pili were expressed in all clone III-1 isolates from Sudan and Sweden but in none of the clone IV-1 isolates (Sudan, 1985).

Antigenic Variation↗

Genomic DNA fingerprinting by pulsed-field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin-resistant Staphylococcus aureus.

In this study, we have compared genomic DNA fingerprintings among isolates of methicillin-resistant Staphylococcus aureus (MRSA) by using pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with SmaI were most suitable for the PFGE separation. SmaI cut genomic DNA into 15 to 20 fragments whose sizes ranged from about 30 to 1,500 kb. Thirty-one distinctive fragment patterns were identified in 111 infecting and colonizing MRSA isolates from six different hospitals in Japan. On the basis of the genomic typing by PFGE, we performed an epidemiological investigation of an outbreak of nosocomial MRSA infections among inpatients in Nagoya University Hospital. Ten types of chromosomal digestion were identified in the 20 strains isolated from 18 infected patients and 1 from colonized hospital personnel. According to the restriction patterns, we found that four types of these strains had caused epidemic infections among 13 patients in the outbreak. Two types (types 1 and 4) of the strains were involved in the death of five patients. The other infections were sporadic. The clarity and polymorphism of the chromosomal digestion patterns enabled us to discriminate between isolates which could not be differentiated by antibiogram or plasmid analysis. Classification of the genomic DNA fingerprinting patterns by PFGE is therefore proposed as a useful method for investigating the source, transmission, and spread of nosocomial MRSA infections.

Biomarkers↗

Application of gas-liquid chromatographic analysis of cellular fatty acids for species identification and typing of coagulase-negative staphylococci.

Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to analyze 264 isolates of coagulase-negative staphylococci, of which 178 were Staphylococcus epidermidis. The presence and amounts of individual fatty acids were determined to generate fatty acid profiles for each of the seven coagulase-negative species tested. The fatty acid profiles were then analyzed by computerized correlation and cluster analysis to calculate mean correlation values between isolates belonging to the same or different species, as well as to establish cluster analysis dendrograms. These data ultimately allowed the clustering of individual samples into species-specific clusters. Species identification by the GLC clustering was highly consistent with species identification by biochemical assays; the results were similar in 92.4% of the cases. The GLC profile correlation analysis was further used to analyze multiple blood isolates from 60 patients in order to determine the usefulness of this methodology in establishing identity, as well as differences, between consecutive patient isolates. The correlation between those multiple S. epidermidis isolates determined to be identical by standard techniques (such as the antibiogram, biotype, and plasmid profile) was significantly (P less than 0.001) higher than that between random isolates of the same species. The correlation coefficient was greater than 97 for 40 (97.6%) of the 41 patients with multiple identical blood isolates, compared with less than 95 in all 19 (100.0%) patients with multiple nonidentical isolates. The successful use of the computerized GLC analysis in this study demonstrated its appropriate application for species identification and typing of coagulase-negative staphylococci.

Bacterial Typing Techniques↗

Aeromonas jandaei (formerly genospecies DNA group 9 A. sobria), a new sucrose-negative species isolated from clinical specimens.

A large numerical taxonomy study conducted in 1988 of 165 mostly clinical Aeromonas strains from diverse geographic sources produced a cluster (S = 84%, SSM) of four sucrose-negative strains that included the DNA definition strain for DNA group 9 A. sobria (CDC 0787-80). These four strains, together with five additional strains received in 1989, were subjected to DNA-DNA hybridization (hydroxyapatite, 32P, 60 and 75 degrees C), and all eight strains were closely related to the ninth labeled DNA group 9 definition strain CDC 0787-80 (73 to 86% relatedness at 60 degrees C and 68 to 80% relatedness at 75 degrees C; percent divergence, 2.0 to 3.5). Type strains and DNA definition strains for all other established Aeromonas species were only 35 to 72% related (60 degrees C) to CDC 0787-80. We propose the name Aeromonas jandaei for this highly related group of nine strains, formerly known as DNA group 9 A. sobria. The type strain was designated ATCC 49568 (CDC 0787-80). The nine strains were examined at 36 degrees C and were found to be resistant to 0/129 (vibriostatic agent) and uniformly positive for oxidase, gas production from glucose, indole, lysine decarboxylase, arginine dihydrolase, o-nitrophenyl-beta-D-galactopyranoside, motility (25 degrees C), nitrate reduction, citrate utilization, hemolysis on sheep blood agar, and growth in Trypticase soy broth with no added NaCl. They all fermented D-glucose, D-mannitol, and mannose but did not ferment sucrose, cellobiose, L-arabinose, inositol, salicin, or D-sorbitol. They were uniformly negative for esculin and urea hydrolysis, elastase production, ornithine decarboxylation, and the string test. The antibiogram of A. jandaei resembled that of other aeromonads (resistance to ampicillin and cephalothin), but it differed from most other aeromonads because of resistance to single dilution of colistin and differed from clinical A. veronii biogroup sorbria (formerly A. sobria) by its nearly uniform resistance to cephalothin. The esculin-, sucrose-, and cellobiose-negative and colistin-resistant profile distinguished A. jandaei from other Aeromonas species. These A. jandaei strains were isolated from blood (two strains), wounds (two strains), diarrheal stools (four strains), and a prawn (one strain). The blood and wound isolates, in particular, suggest that there is a possible clinical significance for this species and justify identification of and further research on this group of motile aeromonads.

Aeromonas↗

Polymicrobic bacteriuria: significant association with bacteremia.

Current clinical practice states that a urine culture yielding three or more isolates must be considered a contaminated specimen and discarded. The validity of this practice was tested by reviewing all patients at the West Haven Veterans Administration Hospital with positive blood cultures for 1 year to determine how many were associated with polymicrobic bacteriuria (two or more urine isolates each present at greater than 10(5) colonies/ml). During 1973 there were 18 patients who had bacteremia associated with a urinary tract infection in which the same organism with the identical antibiogram was found in both the blood and the urine of these 18 patients with monomicrobic bacteremia 11(61%) had polymicrobic bacteriuria, with a mean of an additional 1.75 urinary pathogens present in significant quantities. In these patients with polymicrobic bacteriuria, an indwelling bladder catheter was present for a mean period of 4.4 months before the episode of bacteremia. Only 5 of the 11 patients with polymicrobic bacteriuria had temporally associated urinary tract manipulation. Monomicrobic bacteriuria occurred in seven patients (39%). The mean indwelling bladder catheter time was 0.9 days, and urinary tract manipulation near the time of bacteremia occurred in all seven patients. Prospectively, nine other patients with polymicrobic bacteriuria were recultured by one of us to be certain that appropriate collection and transportation methods were used; the presence of polymicrobic bacteriuria was repeatedly demonstrated in 7(78%). This study illustrates the clinical importance of polymicrobic bacteriuria.

Aged↗

Occurrence of resistance to vibriostatic compound 0/129 in Vibrio cholerae 01 isolated from clinical and environmental samples in Bangladesh.

Fifty-one Vibrio cholerae 01 strains isolated from 734 natural water and plankton samples and 31 rectal swabs were examined. Of these strains, 32 (62.7%) were found to be resistant to vibriostatic compound 0/129. When antibiograms using the antibiotics ampicillin, tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, furoxan, and gentamicin were done, it was observed that there was a correlation of sensitivity to 0/129 with selected antibiotics. Only the Ogawa E1 Tor (72% of strains resistant) and Inaba classical (28% of strains resistant) biotypes of V. cholerae 01 showed resistance to 0/129. On the other hand, all Inaba E1 Tor and Ogawa classical strains were susceptible to 0/129. The 32 0/129-resistant and 19 0/129-sensitive isolates of V. cholerae 01 were tested for the presence of plasmid DNA. Only two strains isolated from the environment were found to carry a plasmid, and they were also found to be resistant to 0/129 and gentamicin. Thus, 0/129 resistance, although more common than previously suspected, is concluded not to be plasmid mediated in the strains tested in this study.

Animals↗

Epidemiologic typing of Staphylococcus aureus by DNA restriction fragment length polymorphisms of rRNA genes: elucidation of the clonal nature of a group of bacteriophage-nontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates.

Analysis of DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) was employed to assist in the epidemiologic investigation of the emergence and spread of ciprofloxacin-resistant Staphylococcus aureus at the Atlanta VA Medical Center because many isolates of interest were nontypeable by phages and harbored few plasmids useful as strain markers. Chromosomal DNAs of selected S. aureus isolates were digested initially with 20 different restriction enzymes. EcoRI appeared to give the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Overall, 15 different ribotypes were seen among the 50 S. aureus isolates studied (7 ribotypes among 13 methicillin-susceptible S. aureus [MSSA] isolates and 9 ribotypes among 37 methicillin-resistant S. aureus [MRSA] isolates). Seven of eight ciprofloxacin-resistant MSSA (CR-MSSA) patient isolates had identical antibiograms, were nontypeable by phages, and had a single 22-MDa plasmid. Six of these seven CR-MSSA isolates had an identical ribotype pattern. Ribotyping distinguished this CR-MSSA strain or clone from MRSA and other MSSA isolates, including nontypeable isolates that contained a 22-MDa plasmid. Five ciprofloxacin-susceptible MSSA isolates studied had five ribotypes; one pattern was identical to the CR-MSSA clone. Twenty-three CR-MRSA isolates recovered from the Atlanta VA Medical Center had four different ribotypes. Ribotyping proved to be a useful molecular epidemiologic tool in the study of S. aureus because it differentiated isolates which were indistinguishable by more traditional methods. In addition, this technique demonstrated that at our institution, ciprofloxacin resistance emerged in multiple strains of MRSA, as opposed to primarily a single strain or clone of MSSA.

Bacterial Typing Techniques↗

E test as susceptibility test and epidemiologic tool for evaluation of Neisseria meningitidis isolates.

The E test (AB Biodisk, Solna, Sweden), a new approach developed to test antimicrobial susceptibility, was compared with the agar dilution method for seven-drug antibiogram analysis of Neisseria meningitidis isolates. The overall E-test quantitative accuracy (+/- 1 log2 dilution) was 93% compared with that of agar dilution testing. The E test was then used to perform the susceptibility tests on a 10-year sample of 102 N. meningitidis isolates, including 5 from a recent epidemic outbreak in the University of Iowa (Iowa City) community. The E test proved to be an efficient methodology for identifying common source clusters of meningococcal disease having resistance to rifampin or sulfonamides. Moreover, the data demonstrated a recent increase in penicillin MICs (MIC for 90% of strains, 0.094 microgram/ml) and an escalation of high-level resistance to trimethoprimsulfamethoxazole (33%) and rifampin (14%). The E test should be considered a simple and accurate susceptibility method for the emerging need to test meningococci and other pathogenic neisserias. Chocolate Mueller-Hinton agar was observed to provide the best support of growth and E-test MIC results that correlated well with results of the reference agar dilution method previously used for neisserias.

Agar↗

Comparison of six typing methods for Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is an important pathogen in the etiology of severe periodontitis. For epidemiological studies on the prevalence of certain pathogenic clones and transmission of this bacterium, adequate typing methods are necessary. The purpose of this study was to compare six different typing methods for A. actinomycetemcomitans. Five reference strains and 27 fresh clinical isolates from periodontitis patients were used. Serotyping showed 12 serotype a strains, 13 type b strains, 6 type c strains, and 1 nontypeable strain. Biotyping on the basis of the fermentation of mannose, mannitol, and xylose resulted in six biotypes. Antibiogram typing was evaluated by measuring the inhibition zones of seven antibiotics in agar diffusion tests. With this method eight main types which could be further differentiated into 15 subtypes were found. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer membrane proteins were similar among all isolates tested. Restriction endonuclease analysis (REA) of whole chromosomal DNA resulted in five main types. These five main types were further differentiated into 24 subtypes on the basis of DNA fragment differences in the high-molecular-weight region. Hybridization of DNA fragments with ribosomal DNA (ribotyping) resulted in 22 to 24 different types, depending on the restriction endonuclease used. Ribotype patterns were easy to interpret and provided an univocal distinction between different strains compared with REA results. When applied to epidemiologically related isolates, all methods were able to discriminate two clonal types among five isolates from five children from one family. We conclude that serotyping, biotyping, and outer membrane patterns were reproducible but had a low discriminatory potential. REA and ribotyping were reproducible and gave the highest number of distinct types. When the DNA typing methodis were compared, all strains tested could be distinguished. These findings confirm the heterogeneity found within the species A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection.

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.

Bacterial Typing Techniques↗

DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex.

Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia.

Bacterial Typing Techniques↗

Clinical and epidemiological investigations of Acinetobacter genomospecies 3 in a neonatal intensive care unit.

A prospective study of Acinetobacter isolates from a neonatal intensive care unit was performed for 24 months. Fifty-six isolates were obtained from 21 patients, and another eight were obtained from environmental specimens. Infection due to Acinetobacter organisms was established for 16 patients, 6 with septicemia, 9 with pneumonia, and 1 with a wound infection. Further investigations were performed with 38 representative isolates. Twenty-nine isolates were identified as unnamed DNA-DNA hybridization group (genomospecies) 3, three were identified as genomospecies 2 (Acinetobacter baumannii), one was identified as genomospecies 5 (Acinetobacter junii), three were identified as genomospecies 14, and two were unclassified. Eight distinguishable protein profiles, coded I through VIII, were found by cell envelope protein electrophoresis. Profile V, a common profile, was observed for 17 isolates that had been recovered from 11 patients and 1 dust specimen. These isolates, all of which belonged to genomospecies 3, had similar antibiograms and biotypes. This study has revealed that genomospecies 3 can be associated with infection and be spread in hospitals.

Acinetobacter↗

Unusually large number of methicillin-resistant Staphylococcus aureus clones in a Portuguese hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hospital de Santa Maria, a 1,300-bed teaching hospital in Lisbon, Portugal, since the mid-1980s with a prevalence of 30% in 1993. A total of 54 MRSA and 93 methicillin-susceptible S. aureus (MSSA) isolates recovered during the first 3 months of 1993 were analyzed for the particular mecA polymorphs and Tn554 attachment sites (in the case of MRSA) and for pulsed-field gel electrophoretic patterns. While all MRSA isolates shared a very similar multidrug resistance antibiogram, molecular methods allowed the identification of an unusually large number of genetic backgrounds (24 different pulsed-field gel electrophoresis patterns in 54 isolates) and three different mecA polymorphs among the MRSA strains. Similar large variation in the genetic backgrounds of MSSA was observed. The most frequent mecA polymorph (mecA type I) was found in association with three different Tn554 patterns. Among the MRSA strains of Hospital Santa Maria, we found two clonal types previously described in Portugal: one corresponding to the dominant clone in an MRSA outbreak at the pediatric ward of the Lisbon Hospital Dona Estefânia and another one identical to the Iberian epidemic clone identified in several Portuguese hospitals and in MRSA outbreaks in Barcelona and Madrid. This suggests that MRSA clones of Hospital de Santa Maria may have been a reservoir for staphylococcal strains over the past decade.

Bacterial Typing Techniques↗

Encephalopathy associated with enteroinvasive Escherichia coli 0144:NM infection.

Central nervous system manifestations typically occur with Shigella gastroenteritis and also in enteric Salmonella and Campylobacter infections. To date no association between enteroinvasive Escherichia coli infection and neurologic symptoms has been described. Two children with diarrhea caused by E. coli 0144:NM had otherwise unexplained encephalopathy manifested by profound stupor in one child and by obtundation and meningismus in the other one. These cases of infection occurred in northern Israel during a period of an unusually high rate of enteric infection caused by this organism. None of the microbiologic properties studied were uniquely attributable to the encephalopathic cases. The two encephalopathic as well as all eight nonencephalopathic isolates studied possessed the 140-MDa invasive plasmid. All 10 isolates examined produced small amounts of cytotoxin by the HeLa cell assay, all were nonmotile, and all had identical antibiograms. Eight of 10 of the isolates had identical plasmid profiles, while 2 isolates (from nonencephalopathic patients) had slightly different plasmid profiles. This is the first report of encephalopathy associated with enteroinvasive E. coli.

Central Nervous System Diseases↗

Comparison of outbreak and nonoutbreak Acinetobacter baumannii strains by genotypic and phenotypic methods.

Thirty-one Acinetobacter baumannii strains, comprising 14 strains from 14 outbreaks in different northwestern European cities and 17 sporadic strains, were compared by investigating various properties of the strains including biotype, antibiogram, cell envelope protein electrophoretic profile, ribotype pattern, and the band pattern generated by a novel genomic fingerprinting method, named AFLP, which is based on the selective amplification of restriction fragments. Results showed that 12 strains from unrelated outbreaks were linked together in two clusters according to their similarities by these typing methods, whereas sporadic strains were more heterogeneous. Outbreak strains appeared to be markedly more resistant to antibiotics than nonoutbreak strains. The uniformity of typing characters in two sets of outbreak strains suggests that strains in each cluster have a common clonal origin.

Acinetobacter↗