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.     

Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of the aroA gene

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 x 10(2) CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.     

Mycobacterium avium complex strains from AIDS patients belong to a homogeneous group ascribed by rRNA typing methods

16S rRNA RFLP analysis of Mycobacterium avium complex (MAC) strains isolated from 25 AIDS patients led to identification of seven ribotypes. The same ribotype was determined for strains from 19 patients with and without disseminated disease. When isolates representing the seven ribotypes were examined for their internal transcribed spacer (ITS) between the 16S and 23S rRNA gene nucleotide sequence, four different sequences, including a new ITS type, were recovered. All isolates with the most common ribotype belonged to the sequevar Mav-B. When MAC strains from AIDS patients were compared by ITS sequencing and ribotyping, a significant degree of homogeneity was observed. The discriminatory level reached with ribotyping might be useful for grouping isolates from different clinical sources.     

An evaluation of intergenic rRNA gene sequence length polymorphism analysis for the identification of Legionella species

There are currently more than 40 species of Legionella and the identification of most of these by standard methods is technically difficult. The aim of this study was to assess the suitability of a previously published PCR-based method of identifying Legionella spp. Intergenic 16S-23S rDNA spacer regions were amplified with primers complementary to conserved regions of the rRNA genes. Following electrophoretic separation of the products, data analyses were performed with the Taxotron software package. Computer-assisted analysis (with an empirically derived error tolerance of 3%) could differentiate only 26 of the 43 strains (representing 43 species), with the remaining 17 species clustering into four groups (group I, comprising 10 species; group II, three species; group III, two species and group IV, two species). Analysis of well-characterised 'non-type' strains of some Legionella spp. (e.g., from type culture collections) resulted in patterns distinct from the corresponding type strain in most cases. Furthermore, recent isolates (identified by conventional methods) were identified by this PCR method to the presumed correct species (or species group) in only a minority of cases. Well characterised strains and recent isolates of Legionella showed heterogeneity within many species. This intra-species variation severely limits the usefulness of the method for the identification of isolates. However, this property may be useful for epidemiological typing within such species.     

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.     

Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria

Broad-range PCR has proven to be useful for the detection of bacteria. A set of broad-range PCR primers directed against conserved regions in the 16S rRNA gene was designed to specifically amplify either gram-positive or gram-negative bacteria. The gram type-specific broad-range PCR correctly classified all 62 pathogenic species tested.     

Genetic heterogeneity of clinical strains of Yersinia enterocolitica traced by ribotyping and relationships between ribotypes, serotypes, and biotypes

A series of 74 Yersinia enterocolitica clinical strains collected in a Spanish region and 10 reference strains, assigned to nine serotypes and five biotypes, were analyzed by ribotyping procedures. Riboprobing, performed separately with HindIII and BglI and using an rrn operon as the probe, generated 13 and 11 ribotypes (discrimination index [DI] = 0.56 and 0.55), respectively. PCR ribotyping, performed with primers complementary to conserved regions of 16S and 23S rRNA genes, generated 13 ribotypes (DI = 0. 56). A combination of data from the three procedures allowed for further discrimination into 17 combined ribotypes (DI = 0.83). The dendrogram obtained by cluster analysis of data from riboprobing indicated a high heterogeneity of the ribosomal DNA regions of the strains under study (similarities between 10 and 92%), which were grouped into three clusters at a similarity level of 0.32. The major cluster included 10 branches, and 7 of these formed a subcluster (similarity coefficient, >83%) represented by strains of serotype O:3 and biotype 2, 3, or 4. The second cluster included four branches, represented by strains belonging to seven non-O:3 serotypes, biotypes 1A and 2, and two of these branches included pyrazinamidase-positive as well as pyrazinamidase-negative strains. The remaining three branches, represented by O:3-biotype 4 strains, formed a third cluster weakly related to the others. Data from this study showed that Y. enterocolitica O:3 organisms assigned to a prevalent and endemic lineage and non-O:3 organisms assigned to three other less-frequent lineages are circulating and causing human disease in the Spanish region under study.     

Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.     

Genomic fingerprinting of Haemophilus somnus by a combination of PCR methods

Twenty-three isolates of Haemophilus somnus were typed by repetitive extragenic palindromic (REP) element-based PCR, enterobacterial repetitive intergenic consensus (ERIC)-based PCR, and PCR ribotyping. A total of 11 types were distinguished by REP-PCR, 13 types were distinguished by ERIC-PCR, and 5 types were distinguished by PCR ribotyping. PCR ribotyping produced a relatively simple pattern and a small number of distinct types, whereas REP- and ERIC-PCR both produced more complex banding patterns but increased the discrimination between strains. Clearly distinguishable profiles were obtained for respiratory and genital isolates of H. somnus by all three typing methods. The results suggest that a combination of all three primer sets provides a high-resolution fingerprinting method for epidemiological studies of H. somnus and for its differentiation from related species.     

Molecular epidemiology of Staphylococcus aureus and Enterococcus faecalis in endophthalmitis

Genomic DNA fingerprint analysis was performed on 39 Staphylococcus aureus and 28 Enterococcus faecalis endophthalmitis isolates collected from multiple clinical centers. Among 21 S. aureus genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the S. aureus clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (P = 0.036 [chi2 test]). In contrast to the S. aureus isolates, the E. faecalis endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.     

A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat

Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.     

Investigation of film curing stages by dielectric analysis and physical characterization

A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.     

Intergenic transcribed spacer PCR ribotyping for differentiation of Saccharomyces species and interspecific hybrids

The taxonomy of the genus Saccharomyces has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Saccharomyces, allows unknown Saccharomyces isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes MaeI and HaeIII could distinguish all species of Saccharomyces from each other, as well as from Candida glabrata, Candida albicans, and Blastomyces dermatitidis. The only exception to this was the inability to distinguish between Saccharomyces bayanus and S. pastorianus (S. carlsbergensis). Furthermore, interspecific hybrids resulting from the mating of sibling species of Saccharomyces were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.     

Inducible amp C beta-lactamase producing gram-negative bacilli from blood stream infections: frequency, antimicrobial susceptibility, and molecular epidemiology in a national surveillance program (SCOPE)

A surveillance study of nosocomial blood stream infections [Surveillance and Control of Pathogens of Epidemiologic Importance (SCOPE)] was conducted during a 14-month period in 1995 to 1996 in approximately 50 American medical centers. Among the 4725 blood stream infections, the etiologic agent was Enterobacter spp. in 230, Citrobacter freundii in 24, and Serratia marcescens in 65. The vast majority of these isolates (89%) had been sent to the University of Iowa including 198 Enterobacter spp. (46 Enterobacter aerogenes, 141 Enterobacter cloacae, 11 other Enterobacter spp.), 23 C. freundii, and 62 S. marcescens. Because these species are capable of producing Amp C beta-lactamase, we examined their susceptibility to 12 broad-spectrum antimicrobial agents. The frequency of resistance to ceftazidime and the molecular epidemiology of ceftazidime-resistant strains was also examined. Among the Enterobacter spp. and C. freundii isolates, resistance to third generation cephalosporins (ceftazidime, ceftriaxone) and broad-spectrum semisynthetic penicillins (piperacillin), with or without an enzyme inhibitor (piperacillin/tazobactam), was high, e.g., 35 to 50%. The S. marcescens isolates were quite susceptible to all agents tested. Both imipenem and cefepime were active against virtually all isolates tested including 84 stably derepressed Amp C-producing ceftazidime-resistant strains of Enterobacter spp. and C. freundii. The overall rank order of activity for the six best agents against these Amp C-producing strains was: imipenem (100% susceptible) > amikacin = cefepime (98.6%) > ciprofloxacin = gentamicin = ofloxacin (93.6 to 94.0%). Molecular typing studies of ceftazidime-resistant E. cloacae using an automated ribotyping system, as well as pulsed-field gel electrophoresis, indicated that although clonal spread of a single strain occurred in some of the medical centers, most of the episodes of bacteremia were caused by patient-unique strains. Control of these resistant organisms will require attention to microbiologic recognition of phenotypes, to infection control practices, and to limiting the overuse of certain extended spectrum beta-lactams.     

The ethics of death-hastening or death-causing palliative analgesic administration to the terminally ill

The Phene Plate (PhP) system is a commercially available typing system based on the measurements of kinetics of selected biochemical reactions of bacteria grown in liquid medium in 96-well microplates. The system uses numerical analysis to identify biochemical phenotypes among the tested strains. In the present study, a set of 16 discriminatory tests were used to differentiate 117 strains of Vibrio cholerae O1 from MExico and Bangladesh. The stability of PhP types of 16 isolates under different storage temperatures and after repeated subcultures were also evaluated. The PhP system had a reproducibility of 95%. Storage either at +4 degrees C or -70 degrees C, did not affect the reactions of the isolates, whereas 4 strains (25%) stored at room temperature and 5 strains (31%) subjected to 30 consecutive subcultures, exhibited minor changes in their biochemical reactions. Endemic isolates of V. cholerae O1 from Bangladesh were more diverse (diversity index = 0.84 to 0.93) than epidemic isolates from Mexico (diversity index = 0.73). Using a collection of 33 heterogeneous isolates of classical biotype of vibrios, PhP typing and ribotyping were compared. PhP typing discriminated more types (n = 23) than ribotyping (n = 5), whereas a combination of both yielded 27 types. The PhP system appears to be a simple, reliable and highly discriminating method for typing of V. cholerae, and may prove especially useful as a first screening method in epidemiological studies of V. cholerae.     

Microbiology of liver and spleen abscesses

To study the aerobic and anaerobic microbiology of liver and spleen abscesses and correlate the results with predisposing factors, potential causes and routes of infection, clinical and laboratory data of 48 patients with liver abscesses and 29 with spleen abscesses treated between 1970 and 1990 were reviewed retrospectively. In liver abscesses, a total of 116 isolates (2.4 isolates/specimen) was obtained; 43 were aerobic and facultative species (0.9 isolates/specimen) and 73 were anaerobic species or microaerophilic streptococci (1.5 isolates/specimen). Aerobic bacteria only were isolated from 12 (25%) abscesses, anaerobic bacteria only from eight (17%), and mixed aerobic and anaerobic bacteria from 28 (58%); polymicrobial infection was present in 38 (79%). The predominant aerobic and facultative isolates were Escherichia coli (11 isolates), Streptococcus group D (8), Klebsiella pneumoniae (5) and Staphylococcus aureus (4). The predominant anaerobes were Peptostreptococcus spp. (18 isolates), Bacteroides spp. (13), Fusobacterium spp. (10), Clostridium spp. (10) and Prevotella spp. (4). There were 12 isolates of micro-aerophilic streptococci. S. aureus and beta-haemolytic streptococci were associated with trauma; Streptococcus group D, K. pneumoniae and Clostridium spp. with biliary disease; and Bacteroides spp. and Clostridium spp. with colonic disease. In splenic abscesses, a total of 56 isolates (1.9 isolates/specimen) was obtained; 23 were aerobic and facultative species (0.8 isolates/specimen), 31 were anaerobic species or micro-aerophilic streptococci (1.1 isolates/specimen) and two were Candida albicans. Aerobic bacteria only were isolated from nine (31%) abscesses, anaerobic bacteria from eight (28%), mixed aerobic and anaerobic bacteria from 10 (34%) and C. albicans in two (7%); polymicrobial infection was present in 16 (55%). The predominant aerobic and facultative isolates were E. coli (5 isolates), Proteus mirabilis (3), Streptococcus group D (3), K. pneumoniae (3) and S. aureus (4). The predominant anaerobes were Peptostreptococcus spp. (11 isolates), Bacteroides spp. (5), Fusobacterium spp. (3) and Clostridium spp. (3). S. aureus, K. pneumoniae and Streptococcus group D were associated with endocarditis, E. coli with urinary tract and abdominal infection, Bacteroides spp. and Clostridium spp. with abdominal infection and Fusobacterium spp. with respiratory infection.     

Characterization of Scedosporium prolificans clinical isolates by randomly amplified polymorphic DNA analysis

Fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate Scedosporium prolificans isolates. A total of 59 arbitrary primers were screened with six unrelated S. prolificans isolates, and a panel of 12 primers was selected. The 12 primers were then used to detect DNA polymorphisms among 17 S. prolificans isolates from 11 patients with systemic S. prolificans infections diagnosed in three hospitals located in geographically different areas of Spain. Eight patients were diagnosed with S. prolificans infection in a single institution over a 6-year period, and two other patients were diagnosed with S. prolificans infection in a different hospital over a 1-year period. No single primer allowed for the discrimination of all the isolates from different patients, but this was possible by combining the RAPD patterns from three primers (UBC 701, AB1.08, and AB1.11 or UBC 701, AB1.08, and UBC 707). However, multiple isolates from the same patient were identical. In this study, we also compared a visual method and a computerized method for the analysis of the RAPD patterns. Both methods were satisfactory and gave few discordances, but given the advantages and disadvantages of each method, both systems should be used together. RAPD analysis provided a fast and economical means of typing S. prolificans isolates, with a high level of discrimination among unrelated isolates. Typing by RAPD analysis confirmed that the S. prolificans infections were epidemiologically unrelated.     

Discrimination of Burkholderia gladioli from other Burkholderia species detectable in cystic fibrosis patients by PCR

A procedure for molecular identification of Burkholderia gladioli is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of B. gladioli from related species (B. cepacia, B. multivorans, B. vietnamiensis, B. mallei, B. pseudomallei, Ralstonia pickettii, and R. eutropha) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis.     

Comparative trial between an ultra-short and long protocol of luteinizing hormone-releasing hormone agonist for ovarian stimulation in in-vitro fertilization

No epidemiological typing system to differentiate among Pseudomonas pseudomallei isolates has been available. Ribotype analysis was developed and used to examine 74 clinical and 10 environmental isolates of P. pseudomallei from Thailand. Six P. pseudomallei ribotypes were identified from restriction fragment polymorphisms of EcoRI chromosomal digests. The predominant ribotype, A, was found in 59 of the isolates examined. By using patterns from hybridizations with SalI, HindIII, and PstI restriction digests, isolates of ribotype A were subdivided into a further five subtypes, giving a total of 10 differentiable P. pseudomallei types. In 23 of 34 melioidosis patients studied, multiple P. pseudomallei isolates were present. In all but one of these patients, a single ribotype of the organism was present. Isolation of two different ribotypes of P. pseudomallei from one patient, one each in sputum and urine, suggests that superinfection may have occurred. The ribotype was shown to be conserved during the course of antibiotic treatments in seven patients studied, although the antibiotic sensitivity patterns in the isolates from these patients varied. The prevalence of subtype A1 in clinical and environmental specimens suggests that this strain may be predominant in this geographical location. These results demonstrate the usefulness of the ribotyping method for epidemiological studies of P. pseudomallei.