Macrorestriction Analysis Research Articles

Long-term clonal lineages within Campylobacter jejuni O:2 strains from different geographical regions and hosts

The genomic diversity of 11 epidemiologically unrelated strains of Campylobacter jejuni (serotype O:2) isolated from different hosts and different geographical regions in Germany over a period of 15 years was studied and results were compared with the reference strain NCTC11168. By flagellin PCR-RFLP typing six fla types were identified, while macrorestriction analysis with three different restriction enzymes revealed almost identical patterns for two human and one bovine strain, even though they were isolated between 1984 and 1996. Interestingly, the PFGE and fla profiles from strain NCTC11168, which was originally isolated in 1977 from an outbreak case in Worcester (UK), were highly similar or even identical to the profiles of these strains. Besides this, mapping of selective genetic markers to the obtained restriction fragments by Southern blot hybridization showed a conserved localization of the investigated sequences in several strains. Our data confirmed considerable degrees of genomic conservation and the occurrence of long-term O:2 serotype-associated clonal lineages in C. jejuni in different geographical regions and hosts.

Clonal Relatedness and Conserved Integron Structures in Epidemiologically Unrelated Pseudomonas aeruginosa Strains Producing the VIM-1 Metallo-β-Lactamase from Different Italian Hospitals

Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-beta-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the bla(VIM-1) determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne bla(VIM-1)-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their bla(VIM-1)-containing integrons.

MOLECULAR CHARACTERIZATION OF BORRELIA ISOLATES FROM TICKS AND MAMMALS FROM THE SOUTHERN UNITED STATES

Fifty Borrelia isolates from ticks and rodents from several geographic regions of the southern United States were analyzed by genomic macrorestriction analysis. Significant genetic diversity was observed among them. These isolates segregated into 4 major clusters and 10 subclusters, which are correlated with the genospecies distribution. Nineteen pulsed-field gel electrophoresis (PFGE) types were recognized among the isolates. The genospecies Borrelia andersonii and Borrelia bissettii consisted of 5 and 2 subclusters, respectively. Two subclusters comprised the Borrelia burgdorferi sensu stricto (s. s.) strains. These results indicated that PFGE is a suitable molecular typing method for B. burgdorferi at both the genospecies and strain levels. Seventeen representative isolates from different PFGE groups were analyzed by restriction fragment length polymorphism (RFLP) and sequence analysis of flaB. Twenty-three AluI, 3 CelII, and 11 DdeI RFLP patterns were found among strains from the B. burgdorferi sensu lato (s. l.) complex and the relapsing fever borreliae complex. Three genospecies in the B. burgdorferi s. l. complex and 1 species in the relapsing fever borreliae complex were recognized. Phylogenetic analysis based on nucleotide sequences of flaB indicated that all the Borrelia strains analyzed here could be divided into 2 parts, i.e., B. burgdorferi s. l. complex and the relapsing fever borreliae complex. The flaB appears to be a useful target gene to screen and identify strains from both B. burgdorferi s. l. and relapsing fever borreliae complexes.

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.

Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry

The molecular biology and epidemiology of 150 avian pathogenic Escherichia coli strains (APEC) isolated from septicemic poultry in Germany was investigated by serotyping, pulsed field gel electrophoresis (PFGE), and polymerase chain reaction (PCR). Only 49.6% of the isolates could be grouped to serogroups O1, O2, and O78. Macrorestriction analyses data revealed two large clonal groups (clusters I and II) among the APEC strains with a similarity of 60.9% to each other. An association between restriction pattern and serogroup or origin of the strains was only present in a few subgroups of each clusters I and II, but was not evident. In contrast, our data revealed distinct combinations of virulence-associated genes in that 51.2% of the O2-strains harboured a combination of the genes fyuA, irp2, iucD, tsh, vat, fimC, and colV and 36.4% of the O78-strains possessed the same gene combination with exception of vat. With 34 different gene combinations the non-O1, -O2, -O78 isolates revealed a higher variability in their virulence gene pattern than O1-, O2-, and O78-strains with 6, 13, and 9 patterns, respectively. Our data indicate only a limited association between the virulence gene pattern and the serogroup of APEC strains and question the sensitivity of O-typing for APEC identification without the application of further diagnostic tools. Although a limited number of APEC clones exist, horizontal gene transfer seems to be common in these pathogens. These findings strengthen further research on the population structure of APEC and may be the reason for the lack of clear definition of this common E. coli pathotype.

Two chimeric chromosomes of Streptomyces coelicolor A3(2) generated by single crossover of the wild-type chromosome and linear plasmid scp1.

Streptomyces coelicolor A3(2) strain 2106 carries a 1.85-Mb linear plasmid, SCP1'-cysD, in addition to a 7.2-Mb linear chromosome. Macrorestriction analysis indicated that both linear DNAs are hybrids of the wild-type chromosome and the linear plasmid SCP1 on each side. Nucleotide sequencing of the fusion junctions revealed no homology between the recombination regions. SCP1'-cysD contains an SCP1 telomere and a chromosomal telomere at each end and therefore does not have terminal inverted repeats. In addition, SCP1'-cysD could not be eliminated from strain 2106 by various mutagenic treatments. Thus, we concluded that both the 7.2-Mb chromosome and SCP1'-cysD are chimeric chromosomes generated by a single crossover of the wild-type chromosome and SCP1. This may be regarded as a model of chromosomal duplication in genome evolution.

High-resolution genotyping of Campylobacter upsaliensis strains originating from three continents.

Ninety-six Campylobacter upsaliensis strains that originated from Australia, Canada, and Europe (Germany) and that were isolated from humans, dogs, and cats were serotyped for their heat-stable surface antigens. All of them were genotyped by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) profiling, and 83 strains were genotyped by macrorestriction analysis with the endonuclease XhoI. Eighty-four percent of the strains belonged to five different serotypes (serotypes OI, OII, OIII, OIV, and OVI), with the proportions of strains in each serotype being comparable among the groups of strains from all three continents. Two serotypes, OIII and OIV, were prevalent at rates of 35 to 40%. Serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. Between 10 and 17.7% of the strains did not react with the available antisera. Analysis of the ERIC-PCR profiles revealed two distinct genotypic clusters, which represented the German and the non-European strains, respectively. XhoI macrorestriction yielded two genotypic clusters; one of them contained 80.2% of the German strains and 34.6% of the non-European strains, and the second cluster consisted of 65.4% of the non-European strains and 19.8% of the German strains. Fourteen strains from all three continents were analyzed for their 16S rRNA gene sequences. Only two minor variations were detected in four of the strains. In conclusion, C. upsaliensis has undergone diverging processes of genome arrangement on different continents during evolution without segregating into different subspecies.

Interhospital transfer of pan-resistant Acinetobacter strains in Johannesburg, South Africa

Background Acinetobacter species infections are increasingly found to cause nosocomial infections, particularly in intensive care units. These pathogens are difficult to eliminate from the hospital environment, and the emergence of multiple-drug-resistant strains complicates patient treatment. In this retrospective study, several strains were analyzed to study the possible spread of pan-resistant strains. Methods Macrorestriction analysis was performed on isolates collected in July 2001 from Johannesburg Hospital and strains collected from a number of hospitals in Johannesburg a year later. Results A strain endemic to Johannesburg Hospital that was cefepime and ceftazidime sensitive in 2001 developed resistance to these antibiotics within 1 year. This and other resistant strains were found to have spread among academic and private hospitals in the area by July 2002. Conclusions The development of resistance is believed to be a response to antibiotic pressure and the spread of resistant strains a result of health care worker and/or patient transfer among hospitals. This snapshot epidemiologic study highlights the need to institute stricter infection control measures to limit the spread of organisms such as Acinetobacter among hospitals.

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis.

To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.

Ambler class A extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals.

This report describes a study carried out to gain baseline information on the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. in Canada. A total of 29,323 E. coli and 5,156 Klebsiella sp. isolates were screened at 12 participating sites. Of these, 505 clinically significant, nonrepeat isolates displaying reduced susceptibility to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. A total of 116 isolates were confirmed to be ESBL producers. PCR and sequence analysis revealed the presence of TEM-11 (n = 1), TEM-12 (n = 1), TEM-29 (n = 1), TEM-52 (n = 4), CTX-M-13 (n = 1), CTX-M-14 (n = 15), CTX-M-15 (n = 11), SHV-2 (n = 2), SHV-2a (n = 12), SHV-5 (n = 6), SHV-12 (n = 45), and SHV-30 (n = 2). Five novel beta-lactamases were identified and designated TEM-115 (n = 2), TEM-120 (n = 1), SHV-40 (n = 2), SHV-41 (n = 4), and SHV-42 (n = 1). In addition, no molecular mechanism was identified for five isolates displaying an ESBL phenotype. Macrorestriction analysis of all ESBL isolates was conducted, as was restriction fragment length polymorphism analysis of plasmids harboring ESBLs. Although a "clonal" distribution of isolates was observed at some individual sites, there was very little evidence suggesting intrahospital spread. In addition, examples of identical or closely related plasmids that were identified at geographically distinct sites across Canada are given. However, there was considerable diversity with respect to plasmid types observed.

Genetic diversity of cell-invasive erythromycin-resistant and -susceptible group A streptococci determined by analysis of the RD2 region of the prtF1 gene.

The RD2 region of the internalization-associated gene prtF1, which encodes the fibronectin-binding repeat domain type 2 of protein F1, plays a crucial role in the entry of group A streptococci (GAS) into epithelial cells. A molecular study of the variability of the RD2 region was carried out with 77 independent Italian GAS, 66 erythromycin resistant (ER) and 11 erythromycin susceptible (ES), which had previously been investigated for the association between erythromycin resistance and ability to enter human respiratory cells. The amplicons obtained from PCR analysis of the RD2 region were consistent with a number of RD2 repeats ranging from one to five, more frequently four (n = 30), three (n = 27), and one (n = 18). A new method to type cell-invasive GAS (RD2 typing) was developed by combining PCR analysis of the RD2 region and restriction analysis of PCR products with endonucleases HaeIII, DdeI, and HinfI. Overall, 10 RD2 types (a to j) were distinguished (all detected among the 66 ER isolates, four detected among the 11 ES isolates). Comparison and correlation of RD2 typing data with the genotype and phenotype of macrolide resistance and with data from PCR M typing and SmaI macrorestriction analysis allowed us to identify 41 different clones (31 among the 66 ER isolates and 10 among the 11 ES isolates). Three major clones accounted for 40% of the isolates (47% of ER strains). Some ES isolates appeared to be related to ER isolates with identical combinations of RD2 type and emm type. While simultaneous use of different typing methods is essential for a thorough investigation of GAS epidemiology, RD2 typing may be especially helpful in typing cell-invasive GAS.

Occurrence of enterotoxin genes and macrorestriction analysis of Staphylococcus aureus isolated from bovine mastitis and bulk-tank milk samples in Italy. An epidemiological study

The goal of the study was to genotypically compare S. aureus isolates from mastitis milk and raw milk to identify the relation between strains and to assess the enterotoxigenicity of the isolates. Eighty-three Staphylococcus aureus isolates recovered from cows and bulk tank milk of five farms in northern Italy were compared genotypically. The genes for the enterotoxins A, D, G and I, but not for B, C, E and H and the toxic shock syndrome toxin 1 (TSST-1), were detected by PCR amplification. Macrorestriction analysis with the restrictions enzyme SmaI revealed 14 pulsed-field gel electrophoresis patterns. These were in part different from each other only in a few fragments and thus displayed a close clonal relation. The results of the present investigation showed that identical or closely related clones seemed to be responsible for the cases of bovine mastitis in the farms investigated and partly responsible for contamination of bulk tank milk.

Two episodes of vancomycin-resistant Enterococcus faecium outbreaks caused by two genetically different clones in a newborn intensive care unit

In 2001 two outbreak episodes (January-March and June-July) caused by vancomycin-resistant E. faecium (VRE) of the VanA-type were observed at a neonatal intensive care unit (NICU) of a university hospital in south-west Germany. To identify the initial source and the route of transmission environmental samples were examined as well as stool samples from patients and the staff. VRE was not found in environmental samples. However, stool samples from 24 hospitalised children tested positive and bacterial clonality was assessed by Sma1-based macro restriction analysis. Furthermore, esp gene and vancomycin resistance gene carriage were examined as well as bacteriocin production. PCR analysis showed that all 24 isolates carried vanA gene cluster, encoding resistance to vancomycin and teicoplanin. However, five of the vanA-positive isolates were resistant to vancomycin but not to teicoplanin. Only these five isolates produced bacteriocin, but in none of the isolates esp gene was detected. PFGE revealed that both outbreaks were caused by two different clones. The patient initiating the first episode, was identified whereas the origin of the second episode remained unknown. From one of the 40 staff stool samples VRE was isolated. This strain was related to the clone of the summer outbreak. In conclusion there were two independent episodes of self limiting VRE outbreaks and transmission on the ward is highly probable.

Persistence of a clone of glycopeptide-resistant Enterococcus faecalis among patients in an intensive care unit of a Greek hospital.

To investigate an outbreak of glycopeptide-resistant Enterococcus faecalis (GREF) in the intensive care unit (ICU) of 'G. Gennimatas' General Hospital, Athens, Greece. Between August 2000 and November 2001, 20 highly GREF isolates were recovered from severe infections of separate patients in the ICU. The isolates were tested by PCR, PFGE, mating experiments and plasmid analysis. All isolates carried the vanA gene. Nineteen isolates fitted to one clone by macrorestriction analysis with four subclones being consecutively detected. Each subclone seemed to predominate for a specific time period. Additionally, four GREF isolates related to the ICU clone were recovered from other wards of the hospital. Our findings indicate that a monoclonal GREF outbreak persisted for more than 1 year in a large Greek hospital. The rate of GREF isolation declined after the application of infection control measures.

Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs.

The aim of this study was to analyse florfenicol-resistant Escherichia coli isolates from pigs for the genetic basis of florfenicol resistance, and to compare these data with those previously determined for E. coli isolates from cattle and poultry. Fourteen porcine E. coli isolates were included in this study and subjected to serotyping, plasmid profiling and macrorestriction analysis. MICs of florfenicol were determined by broth microdilution. The presence of the gene floR was confirmed by hybridization and PCR analysis. Transformation experiments were conducted to isolate florfenicol resistance plasmids. The floR region of a florfenicol resistance plasmid was cloned and sequenced. All florfenicol-resistant E. coli isolates exhibited MICs of florfenicol >128 mg/L and carried the floR gene. A single isolate had a floR-carrying plasmid of approximately 35 kb, designated pMBSF1. Sequence analysis identified the floR gene flanked by truncated transposase genes. Moreover, a truncated copy of Tn5393 with complete streptomycin resistance genes strA and strB was found upstream of the floR gene of pMBSF1. Chromosomally resistant E. coli isolates, which shared the same BlnI macrorestriction pattern, differed in their floR hybridization patterns. The plasmid pMBSF1 is the smallest floR-carrying plasmid reported to date. Its floR region differed from those previously found in E. coli isolates from cattle. Variations in the RFLPs of chromosomal EcoRI fragments carrying floR in isolates that had the same macrorestriction pattern might suggest variable chromosomal integration sites.

Extended-spectrum beta-lactamases in Klebsiella spp and Escherichia coli obtained in a Brazilian teaching hospital: detection, prevalence and molecular typing

His study was performed to compare the methods of detection and to estimate the prevalence of extendedspectrum β-lactamases (ESBL) among Klebsiella spp and E. coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K. pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clinicas de Porto Alegre, using Kirby-Bauer zone diameter (KB), double-disk diffusion (DD), breakpoint for ceftazidime (MIC CAZ), increased zone diameter with clavulanate (CAZ/CAC) and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC). Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E. coli), contrasting with all the other methods (p < 0.05). The confirmatory methods (DD, MIC CAZ, CAZ/CAC and MIC CAZ/CAC) showed a range of ESBL production from 8 to 13% for E. coli and from 33 to 40% for Klebsiella species. Therefore, the KB method was useful only as a screening method as it provided several false positive results. Molecular typing of 17 ESBL K. pneumoniae indicated that the isolates had no clonal relation. We found a good correlation among the confirmatory methods for ESBL detection although the methods which evaluate inhibition of the β-lactamase by clavulanate appeared to be more specific. The high prevalence of ESBL Klebsiella in our hospital is probably due to individual selection of resistant strains rather than the transmission of a common strain.

Characterization of the natural population of Bartonella henselae by multilocus sequence typing.

Investigations of the population genetics of Bartonella henselae have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of B. henselae by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among B. henselae strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.

Tetracycline Resistance in Staphylococci from Free‐living Rodents and Insectivores

One hundred and fifty-eight staphylococcal strains isolated from wild rodents and insectivores were analysed for plasmid-borne resistance to tetracycline (Tc). Only 10 isolates, six Staphylococcus saprophyticus isolates and single isolates of S. xylosus, S. equorum, S. warneri and S. cohnii subsp. cohnii carried a Tc resistance plasmid of approximately 4.4 kb as confirmed by protoplast transformation. All 10 plasmids harboured a Tc resistance gene of hybridization class K [tet(K)] as confirmed by polymerase chain reaction (PCR). The plasmid was assigned to the pT181 family as it revealed a high degree of restriction map homology to pT181 and other members of this family. Macrorestriction analysis with the enzyme SmaI showed that three of the six isolates identified as S. saprophyticus shared the same pulsed-field gel electrophoresis (PFGE) pattern.

Macrococcus brunensis sp. nov., Macrococcus hajekii sp. nov. and Macrococcus lamae sp. nov., from the skin of llamas.

Eight strains of Gram-positive, catalase- and oxidase-positive cocci were isolated from the skin of llamas (Lama glama L.) and characterized using a polyphasic approach. These strains were assigned to the genus Macrococcus on the basis of their phenotypic properties (resistance to bacitracin and sensitivity to furazolidone) and DNA base content (40-42 mol% G+C). Phylogenetic analysis based on 16S rDNA confirmed that the strains are members of the genus MACROCOCCUS: They differed from all hitherto described macrococcal species in their production of phosphatase and reduction of nitrate (most strains) and the inability to produce acid from glycerol or to grow in 7.5 % NaCl. Ribotyping (EcoRI), macrorestriction analysis (XbaI) and fatty acid methyl ester analysis divided the strains from llamas into three stable clusters. Moreover, ribotyping differentiated the strains analysed not only from previously described macrococcal species but also from oxidase-positive staphylococci. DNA-DNA hybridization confirmed that the three clusters represent separate genomic groups (similarity values<54 %). All the results showed that the strains represent three novel species, for which the names Macrococcus hajekii sp. nov. (type strain CCM 4809(T)=LMG 21711(T)), Macrococcus brunensis sp. nov. (type strain CCM 4811(T)=LMG 21712(T)) and Macrococcus lamae sp. nov. (type strain CCM 4815(T)=LMG 21713(T)) are proposed.

Determination of epidemiological relationships of Streptococcus agalactiae isolated from bovine mastitis

In the present study 79 streptococcal cultures isolated from subclinical mastitis of 54 cows from seven dairy farms (A–G) in Hesse, Germany, were comparatively investigated using conventional and molecular methods. The isolates could be identified as Streptococcus agalactiae, belonging to Lancefield’s serological group B by determination of cultural, biochemical and serological properties and by polymerase chain reaction (PCR)-mediated amplification of species-specific parts of the 16S ribosomal DNA, the 16S–23S rDNA intergenic spacer region and the CAMP factor gene cfb. The investigated group B streptococci were further characterized serologically for specific polysaccharide and protein antigens. Serotyping the isolates revealed a predominance of surface protein antigen X, either alone or in combination with polysaccharide antigen Ia. This could be observed for 39 isolates of farms A, B and C. Six group B streptococci from farm E displayed the serotype pattern III/Rib, two isolates from farm G showed the serotype pattern Ib/cα. The remaining cultures from farms D and F ( n=32) were non-typable. The occurrence of protein Rib could be confirmed by PCR amplification of the gene rib. The two isolates with serotype pattern Ib/cα also reacted positively for the cβ-encoding gene bag. Additional properties which allowed a phenotypic characterization of the S. agalactiae were the degree of pigmentation, growth properties in fluid media and soft agar, the surface hydrophobicity, the ability to hemagglutinate rabbit erythrocytes and their resistance reactions to tetracycline and minocycline. The isolates of the seven farms showed identical or almost identical characteristics. The 79 group B streptococci were additionally investigated by macrorestriction analysis of their chromosomal DNA using the restriction endonucleases SmaI, ApaI and SalI. The restriction patterns obtained by pulsed-field gel electrophoresis displayed identical or closely related patterns for the cultures of the various farms. The pheno- and genotypic characteristics of the 79 group B streptococci of the present study revealed that a single S. agalactiae strain or at least closely related subtypes of this strain were responsible for the mastitis situation of the seven farms.