HindIII Ribotypes Research Articles

A 63 kb genomic resistance island found in a multidrug-resistant Acinetobacter baumannii isolate of European clone I from 1977

Multidrug-resistant Acinetobacter strain HK302 was isolated from an outbreak of nosocomial infections in Switzerland in 1977. The aim of the present study was to assess whether this archive strain belongs to one of the known international clonal lineages of Acinetobacter baumannii and whether it harbours a genomic structure related to the AbaR1-like resistance islands. Multilocus sequence typing (MLST) and HindIII ribotyping were used to determine the taxonomic position of HK302 at the species and subspecies (clonal) levels. The position and structure of the putative resistance island were investigated by AbaR1-based PCR mapping followed by restriction analysis and partial sequencing of amplicons. A. baumannii AYE harbouring AbaR1 was used as a positive control for PCR mapping. The MLST allelic profile (1-1-1-1-5-1-1) and HindIII ribotype of HK302 were typical of A. baumannii European (EU) clone I. In addition, an AbaR1-related region inserted into the ATPase gene at the same position as AbaR1 was found in HK302. PCR mapping and partial sequencing revealed that this region is structurally congruent with AbaR3, a 63.4 kb island described in an A. baumannii isolate from 2004. A. baumannii HK302 belongs to EU clone I and harbours an AbaR3-like island related to resistance islands described in EU clone I strains. Our findings suggest that variants of these sophisticated genomic structures already existed in A. baumannii in the late 1970s.

Occurance ofStaphylococcus nepalensisstrains in different sources including human clinical material

Five isolates of coagulase-negative staphylococci were obtained from human urine, the gastrointestinal tract of squirrel monkeys, pig skin and from the environment. All key biochemical characteristics of the tested strains corresponded with the description of Staphylococcus xylosus species. However, partial 16S rRNA gene sequences obtained from analysed strains corresponded with those of Staphylococcus nepalensis reference strains, except for two strains which differed in one residue. Ribotyping with EcoRI and HindIII restriction enzymes, whole cell protein profile analysis performed by SDS-PAGE and SmaI macrorestriction analysis were used for more precise characterization and identification of the analysed strains. Obtained results showed that EcoRI and HindIII ribotyping and whole cell protein fingerprinting are suitable and reliable methods for the differentiation of S. nepalensis strains from the other novobiocin resistant staphylococci, whereas macrorestriction analysis was found to be a good tool for strain typing. The isolation of S. nepalensis is sporadic, and according to our best knowledge this study is the first report of the occurrence of this species in human clinical material as well as in other sources.

Diversity of Weissella viridescens strains associated with “Morcilla de Burgos”

Unidentified heterofermentative lactic acid bacterium (LAB) isolates detected earlier in vacuum or modified atmosphere (MA) packaged “Morcilla de Burgos”, a Spanish cooked meat product, were identified in the present study. In the previous study, these strains had been studied by numerical analysis of HindIII RFLP- and phenotyping. However, neither of these methods had resulted in species identification. Even these unknown strains clustered together in the numerical analysis of the RFLP patterns, none of the typically meat-associated LAB type strains had clustered together with them. The phenotyping was not more useful; according to the criteria used these strains with similar HindIII ribopatterns should have been divided into four different species in three different genera. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of EcoRI and HindIII ribopatterns, and DNA–DNA hybridization experiments were done. The 16S rRNA genes sequences of three isolates possessed the highest similarities with the corresponding sequences of Weissella species and in the phylogenetic analysis Weissella viridescens was their closest phylogenetic neighbor. However, in the numerical analysis of either HindIII or EcoRI ribopatterns the W. viridescens type strain did not cluster with these isolates. Nevertheless, the high (from 75% to 83%) DNA–DNA reassociation values between the strains studied and the W. viridescens type strain confirmed that these strains belong to the latter species. The somewhat divergent results of the pheno- and HindIII ribotyping indicate that W. viridescens is far more heterogeneous species than previously reported.

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed

The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.

Delineation of the endemic and sporadic methicillin-resistant Staphylococcus aureus clones in a Czech hospital.

The aim of this study was to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected between September, 2001, and February, 2003, at the regional hospital of Nový Jicín, Czech Republic. The isolates were characterized by susceptibility tests, HindIII ribotyping, and pulsed-field gel electrophoresis. Representatives of each clonal type were analyzed by multilocus sequence typing and staphylococcal cassette chromosome mec (SCCmec) typing. The prevalence of the most important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aac6'-aph2", aph3', and ant4') resistance genes was evaluated as well. Our results document the existence of two international MRSA clones: (1) the Iberian clone (ST247:SCCmec IA:PFGE A:ribotype H2), endemic in the hospital and associated to a single multiresistant phenotype; and (2) clone EMRSA-15 (ST22:SCCmec IV:PFGE H-ribotype H7), appearing in the beginning of 2002 and associated with three phenotypes. These two clones could be distinguished by antibiogram, distribution of macrolide and aminoglycoside resistance genes (ermA, aac6'-aph2", ant4' versus ermC and msrA in a few isolates), production of beta-lactamase, and presence of enterotoxin A (in the Iberian clone).

Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples.

A taxonomic study was conducted to clarify the relationships of two bacterial populations belonging to the genus Weissella. A total of 39 strains originating mainly from Malaysian foods (22 strains) and clinical samples from humans (9 strains) and animals (6 strains) were analysed using a polyphasic taxonomic approach. The methods included classical phenotyping, whole-cell protein electrophoresis, 16S and 23S rDNA RFLP (ribotyping), determination of 16S rDNA sequence homologies and DNA-DNA reassociation levels. Based on the results, the strains were considered to represent two different species, Weissella confusa and a novel Weissella species, for which the name Weissella cibaria sp. nov. is proposed. Weisella confusa possessed the highest 16S rDNA sequence similarity to Weisella cibaria, but the DNA-DNA reassociation experiment showed hybridization levels below 49% between the strains studied. The numerical analyses of Weisella confusa and Weisella cibaria strains did not reveal any specific clustering with respect to the origin of the strains. Based on whole-cell protein electrophoresis, and ClaI and HindIII ribotyping patterns, food and clinical isolates were randomly located in the two species-specific clusters obtained.

The value of rRNA gene restriction site polymorphism analysis for delineating taxa in the genus Staphylococcus.

A total of 101 staphylococcal strains were ribotyped using EcoRI and HindIII as restriction enzymes and plasmid pBA2 as the rDNA probe. Isolates from 10 newly described staphylococcal taxa were among those examined. All the ribotypes were added to our database, Staph DB, which now contains the sizes of the bands of 135 EcoRI and 120 HindIII ribotypes from 408 strains belonging to 42 staphylococcal taxa. The relatedness of ribotypes was evaluated by using the Dice coefficient. The ribotypes, and thus the strains, were clustered by the unweighted pair group method with averages (UPGMA). Separation into clusters correlated well with the delineation of the staphylococcal species but not with that of the different subspecies. No discrimination was possible between Staphylococcus vitulinus and Staphylococcus pulvereri. Ecovar-specific groups were evident within Staphylococcus intermedius and Staphylococcus hyicus. The data increase the usefulness of rRNA gene restriction site polymorphism analysis for staphylococcal taxonomy.

Ribotyping as an identification tool for Clostridium botulinum strains causing human botulism

Ribotyping was used for characterisation of 68 Clostridium botulinum strains and five related Clostridium species to determine the applicability of this method for identification of species causing human botulism. Thirteen restriction enzymes were initially tested for suitability for ribotyping of C. botulinum, of which EcoRI and HindIII were selected. Both enzymes clearly differentiated between proteolytic (group I) and a nonproteolytic (group II) strains of C. botulinum, and can be recommended for Group/species identification. Using a commercial software package (GelCompar), a numerical analysis of the discriminatory abilities of EcoRI and HindIII ribotyping within and between the two C. botulinum groups was performed. EcoRI had the higher discriminatory index (0.982), but the ribopatterns generated with group II strains were partly muddled and difficult to interpret. All HindIII ribopatterns were easy to analyse and the discriminatory index for all strains was almost equally high (0.954), whereas this enzyme did not discriminate well between group I isolates. The Clostridium strains diverged at 35±13% (mean±standard deviation) Dice similarity in dendrograms based on cluster analysis of the ribotyping results. These findings are in good agreement with taxonomical ribotyping studies with other bacterial genera, indicating that ribotyping is a highly suitable method for C. botulinum species identification.

Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark

The aim of the present study was to evaluate capsular-typing, plasmid-profiling, phage-typing and ribotyping for epidemiological studies of toxin-producing Pasteurella multocida ssp. multocida in Denmark. The evaluation of methods was based on 68 strains from nasal swabs and 14 strains from pneumonic lungs. Strains from lungs were all of capsular Type A, whereas strains from nasal swabs were of both capsular Types A and D. Only 9% of the strains contained plasmids, which could not be associated with antibiotic resistance. Phage-typing divided 61% of strains into 10 groups, while 39% were non-typable. CfoI ribotyping divided strains into four groups of which one type contained 94% of isolates. HindIII ribotyping divided strains into 18 types. A total of 18 strains from The Netherlands, UK and USA were subjected to HindIII ribotyping, resulting in 13 types of which six were identical to ribotypes of Danish strains. Phage-typing of isolates from an outbreak of atrophic rhinitis involving six herds in 1985 showed the existence of an epidemic strain. This type was recognised in the herd suspected of being the source of the infections and in four of the five infected herds. These findings were supported by HindIII ribotyping, as 85% of isolates from all herds were assigned to one ribotype. In conclusion, HindIII ribotyping seems to represent a useful tool for epidemiological studies of toxigenic P. multocida ssp. multocida.

Genomic relationships of Actinobacillus pleuropneumoniae serotype 2 strains evaluated by ribotyping, sequence analysis of ribosomal intergenic regions, and pulsed-field gel electrophoresis.

The aim of the present study was to examine the genomic relationship among 112 Actinobacillus pleuropneumoniae serotype 2 strains obtained throughout Europe and North America. HindIII ribotyping of the strains resulted in five ribotypes of high similarity (87-98%). Sequence analysis of the ribosomal intergenic region of strains representing each ribotype and each country showed no differences. A common ribotype was further characterized by PFGE of 12 strains representing all countries. The resultant five PFGE patterns of European strains showed a similarity of more than 91%, to which the two PFGE patterns of the Canadian strains showed a similarity of 84-86%. In conclusion, strains of A. pleuropneumoniae serotype 2 seem to be clonally related.

Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark

The aim of the present study was to evaluate ribotyping as an epidemiological tool for Actinobacillus pleuropneumoniae and apply the method in studies of A. pleuropneumoniae infections in Danish pig herds. The evaluation of ribotyping was based on the 13 international reference strains and 106 epidemiologically unrelated Danish field strains representing the nine serotypes of biotype 1 (1, 2, 5A/B, 6, 7, 8, 10, 12, and K2:O7) and one serotype 14 of biotype 2. Enzymes CfoI and HindIII were chosen for generation of ribotype patterns. Ribotyping of the reference strains resulted in 10 CfoI types and 11 HindIII types. Ribotyping of the Danish strains resulted in 17 different CfoI ribotypes and 24 different HindIII ribotypes. Combining HindIII- and CfoI-ribotyping divided the Danish strains into 26 different types. The stability, reproducibility and typability of ribotype patterns were good, and the discriminatory power was between 0.85–0.89. The relatively low discriminatory power was caused by four predominant types, containing 61% of the isolates. The typing system was applied in studies of routes of infection of specific pathogen-free (SPF) pig herds and included 112 strains of A. pleuropneumoniae. Airborne transmission from neighboring conventional pig farms was investigated in 12 cases of infected SPF herds. Transmission via vehicles transporting pigs between SPF herds was investigated in nine cases while transmission by trading of pigs between SPF herds was investigated in two cases. Serotype 2 was isolated from all SPF herds included in this study, except one, emphasizing the high prevalence of this serotype in Denmark. By ribotyping, airborne transmission was indicated in five of 12 cases, transmission via pig transporting vehicle was indicated in six of nine cases, and transmission via trading was indicated in one of two cases. In many cases findings of predominant ribotypes made interpretations of suspected routes of transmission difficult. The relationship of strains based on ribotypes was calculated using Dices coefficient and clustered by UPGMA. HindIII ribotypes of serotype 2 strains were closely related, though only showing 43% similarity to HindIII ribotypes of remaining serotypes.

Vibrio furnissii isolated from humans in Peru: a possible human pathogen?

During a cholera surveillance programme, Vibrio furnissii was isolated in late January and early February 1994 from stool samples collected from 14 persons of whom six had diarrhoea. The remaining eight persons were healthy family members or neighbours to cholera cases. No common source of infection was found. Strains isolated from stool samples each showed typical biochemical reactions of V. furnissii including gas production. Each isolate, except one, agglutinated O-antisera yielding a total of eight different serotypes. Most isolates were sensitive to 10 antibiotics tested, except to ampicillin and the vibriostatic agent O/129 (10 micrograms). Eight of 14 (57%) strains carried plasmids in the size range 2.6-88 kb, however, no correlation was found between antibiotic susceptibility patterns and plasmid content. Altogether, seven closely related HindIII ribotypes were observed among the 14 V. furnissii isolates studied. V. furnissii strains isolated from family members and other persons living close together often showed different ribotypes suggesting that the isolation was not associated with neighbourhood. Serotyping, plasmid profiling and ribotyping revealed a high strain diversity within V. furnissii, however, the importance of V. furnissii as an enteric pathogen remains to be elucidated.

Genotypic and phenotypic diversity of Streptococcus dysgalactiae strains isolated from clinical and subclinical cases of bovine mastitis

This study was conducted to characterize Streptococcus dysgalactiae isolated from cases of bovine mastitis. One hundred strains from 100 dairy herds in Denmark, 29 strains from 29 dairy herds in Pennsylvania, USA, 7 reference strains and 18 strains consisting of 9 paired strains from 9 quarters were investigated using biotyping (utilization of tagatose and sorbitol) and HindIII ribotyping. Four different biotypes were observed. Only 34 of the 100 Danish strains utilized either tagatose or sorbitol, 18 utilized tagatose, 15 sorbitol and 1 both tagatose and sorbitol. In contrast, among the American strains one (3.4%) did neither utilize tagatose or sorbitol, twenty (69.0%) utilized tagatose, four (13.8%) sorbitol and four (13.8%) both tagatose and sorbitol. HindIII ribotyping produced 15 different types among all the strains, 9 different types were found among the Danish strains and 8 types among the American strains. Five types were observed among both the Danish and the American strains. The 7 reference strains were assigned to 5 different ribotypes. Three type strains (ATCC 27957, NCTC 4670, NCTC 4335) were assigned to a type that was present among both the Danish and the American strains. Ribotyping was the most discriminatory typing method. Agreement between ribo- and biotyping was observed, and especially pronounced among the Danish strains. The 9 paired strains were in each case found to have identical ribo- and biotype.

Ribotyping of Nosocomial Methicillin-Resistant Staphylococcus aureus Isolates From a Canadian Hospital

To evaluate the clonality of methicillin-resistant Staphylococcus aureus (MRSA) strains among hospitalized patients. University-affiliated, 465-bed tertiary-care teaching hospital with adjacent cancer clinic in Hamilton, Ontario, Canada. Thirty-five colonized and 30 infected patients from January 2, 1992, through August 31, 1993, were investigated retrospectively. Analysis by restriction fragment-length polymorphisms of ribosomal RNA genes (ie, ribotyping) of 103 nosocomial isolates of MRSA from these 65 patients and of 25 selected unrelated strains was completed. Ribotyping results were compared with the phage typing data obtained prospectively during the course of prospective MRSA surveillance. HindIII ribotyping was more discriminating than phage typing when epidemiologically unrelated strains were differentiated by these methods (19 different ribotypes versus 14 page types; P < .005). Two early index cases were identified. Isolates from the index cases were two different strains, identified by ribotyping analysis as ribotype A (clonal group 1) and ribotype B (clonal group 2), respectively. These two ribotypes were not found when typing the unrelated control strains. Thirty-six colonized and infected patients (55%) had clonal group 1 isolates, and 20 (31%) had clonal group 2 isolates. These two clones emerged in the hospital in January and February 1992 and dominated the entire investigated period. There also were six patients with an additional clonal group (group 4) that emerged and disappeared in the second quarter of 1993. This study highlights the utility of ribotyping in investigating nosocomial MRSA. Three MRSA clones caused nosocomial colonization or infection in patients at this hospital. Two of these MRSA clones, once introduced, were maintained among our patients throughout the study period.

Molecular characterization of Haemophilus ducreyi by ribosomal DNA fingerprinting.

Intraspecies genotypic heterogeneity among Haemophilus ducreyi isolates was examined by using genomic fingerprints with rRNA from Escherichia coli as a probe. DNA from 44 isolates of H. ducreyi was digested by restriction endonuclease HincII or HindIII, separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with 32P-labeled 16S and 23S rRNA. HincII digests yielded four hybridization patterns (ribotypes), whereas HindIII digests yielded eight ribotypes. Four HincII and five HindIII ribotypes were observed among 14 H. ducreyi isolates collected within a period of 1 month in Kenya, where chancroid is endemic. In contrast, one HincII and two HindIII ribotypes were observed among 28 isolates collected during the Orange County, Calif., chancroid epidemic that occurred in 1981 and 1982. The plasmid content, in conjunction with ribotyping, provided additional differentiation among some isolates of H. ducreyi. This study demonstrates that ribotyping of H. ducreyi may be used to study the epidemiology of chancroid.