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PubMed2005

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

Animals↗
PubMed2004

Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia.

Increasing reports of the appearance of novel nonmultiresistant methicillin-resistant Staphylococcus aureus MRSA (MRSA) strains in the community and of the spread of hospital MRSA strains into the community are cause for public health concern. We conducted two national surveys of unique isolates of S. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. A total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were MRSA. Approximately 54% of the MRSA isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. The majority of multiresistant MRSA isolates in both surveys belonged to two strains (strains AUS-2 and AUS-3), as determined by pulsed-field gel electrophoresis (PFGE) and resistogram typing. The 3 AUS-2 isolates and 10 of the 11 AUS-3 isolates selected for multilocus sequence typing (MLST) and staphylococcal chromosomal cassette mec (SCCmec) analysis were ST239-MRSA-III (where ST is the sequence type) and thus belonged to the same clone as the eastern Australian MRSA strain of the 1980s, which spread internationally. Four predominant clones of novel nonmultiresistant MRSA were identified by PFGE, MLST, and SCCmec analysis: ST22-MRSA-IV (strain EMRSA-15), ST1-MRSA-IV (strain WA-1), ST30-MRSA-IV (strain SWP), and ST93-MRSA-IV (strain Queensland). The last three clones are associated with community acquisition. A total of 14 STs were identified in the surveys, including six unique clones of novel nonmultiresistant MRSA, namely, STs 73, 93, 129, 75, and 80slv and a new ST. SCCmec types IV and V were present in diverse genetic backgrounds. These findings provide support for the acquisition of SCCmec by multiple lineages of S. aureus. They also confirm that both hospital and community strains of MRSA are now common in nonhospitalized patients throughout Australia.

Anti-Bacterial Agents↗
PubMed2005

Use of variable-number tandem repeats to examine genetic diversity of Neisseria meningitidis.

Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of Neisseria meningitidis and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107 N. meningitidis isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s.

Bacterial Typing Techniques↗
PubMed2003

Clonal distribution of invasive pneumococcal isolates from children and selected adults in the United States prior to 7-valent conjugate vaccine introduction.

Theseven-valent pneumococcal conjugated polysaccharide vaccine PC7V was licensed for use among children in 2000. Since 90 serotypes of pneumococci exist, an increase in nonvaccine serotypes could occur through immune selection for capsular type switching. Eleven hundred sixty-eight invasive isolates (24 serotypes), recovered primarily from pediatric patients (855 isolates = 73%) and 22 reference strains of known multilocus sequence types (STs) were subjected to macrorestriction profiling (pulsed-field gel electrophoresis [PFGE]). The correlation of 187 ST results (including 49 newly discovered STs) with the PFGE data assigned 1,042 (89.2%) study isolates to 46 defined clonal complexes or genetic lineages based on related multilocus STs (BURST). Seventeen clonal complexes were represented by 2 to 10 related allelic profiles (STs), while 33 lineages (including reference strains) consisted of single STs with 4 or fewer allelic identities to other STs found in the study. Expansion of the BURST analysis to a global analysis of all known pneumococcal STs (as of 27 November 2002) reduced the number of single ST lineages from 33 to 8, and the number of multi-ST clonal complexes was reduced from 17 to 13. In this work, we established the basic genetic structure within individual serotypes prior to PC7V use. The resultant database will be useful for detecting potential selective effects of this vaccine in postvaccine surveillance.

Adult↗
PubMed2006

Use of a multilocus variable-number tandem repeat analysis method for molecular subtyping and phylogenetic analysis of Neisseria meningitidis isolates.

BACKGROUND: The multilocus variable-number tandem repeat (VNTR) analysis (MLVA) technique has been developed for fine typing of many bacterial species. The genomic sequences of Neisseria meningitidis strains Z2491, MC58 and FAM18 have been available for searching potential VNTR loci by computer software. In this study, we developed and evaluated a MLVA method for molecular subtyping and phylogenetic analysis of N. meningitidis strains. RESULTS: A total of 12 VNTR loci were identified for subtyping and phylogenetic analysis of 100 N. meningitidis isolates, which had previously been characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The number of alleles ranges from 3 to 40 for the 12 VNTR loci; theoretically, the numbers of alleles can generate more than 5 x 1011 MLVA types. In total, 93 MLVA types were identified in the 100 isolates, indicating that MLVA is powerful in discriminating N. meningitidis strains. In phylogenetic analysis with the minimal spanning tree method, clonal relationships, established with MLVA types, agreed well with those built with ST types. CONCLUSION: Our study indicates that the MLVA method has a higher degree of resolution than PFGE in discriminating N. meningitidis isolates and may be a useful tool for phylogenetic studies of strains evolving over different time scales.

Bacterial Typing Techniques↗
PubMed2005

Molecular characterization of pneumococci with efflux-mediated erythromycin resistance and identification of a novel mef gene subclass, mef(I).

The molecular genetics of macrolide resistance were analyzed in 49 clinical pneumococci (including an "atypical" bile-insoluble strain currently assigned to the new species Streptococcus pseudopneumoniae) with efflux-mediated erythromycin resistance (M phenotype). All test strains had the mef gene, identified as mef(A) in 30 isolates and mef(E) in 19 isolates (including the S. pseudopneumoniae strain) on the basis of PCR-restriction fragment length polymorphism analysis. Twenty-eight of the 30 mef(A) isolates shared a pulsed-field gel electrophoresis (PFGE) type corresponding to the England14-9 clone. Of those isolates, 27 (20 belonging to serotype 14) yielded multilocus sequence type ST9, and one isolate yielded a new sequence type. The remaining two mef(A) isolates had different PFGE types and yielded an ST9 type and a new sequence type. Far greater heterogeneity was displayed by the 19 mef(E) isolates, which fell into 11 PFGE types, 12 serotypes (though not serotype 14), and 12 sequence types (including two new ones and an undetermined type for the S. pseudopneumoniae strain). In all mef(A) pneumococci, the mef element was a regular Tn1207.1 transposon, whereas of the mef(E) isolates, 17 carried the mega element and 2 exhibited a previously unreported organization, with no PCR evidence of the other open reading frames of mega. The mef gene of these two isolates, which did not match with the mef(E) gene of the mega element (93.6% homology) and which exhibited comparable homology (91.4%) to the mef(A) gene of the Tn1207.1 transposon, was identified as a novel mef gene variant and was designated mef(I). While penicillin-nonsusceptible isolates (three resistant isolates and one intermediate isolate) were all mef(E) strains, tetracycline resistance was also detected in three mef(A) isolates, due to the tet(M) gene carried by a Tn916-like transposon. A similar mechanism accounted for resistance in four of the five tetracycline-resistant isolates carrying mef(E), in three of which mega was inserted in the Tn916-like transposon, giving rise to the composite element Tn2009. In the fifth mef(E)-positive tetracycline-resistant isolate (the S. pseudopneumoniae strain), tetracycline resistance was due to the presence of the tet(O) gene, apparently unlinked to mef(E).

Amino Acid Sequence↗
PubMed2003

Molecular evidence for dissemination of unique Campylobacter jejuni clones in Curaçao, Netherlands Antilles.

Campylobacter jejuni isolates (n = 234) associated with gastroenteritis and the Guillain-Barré syndrome (GBS) in the island of Curaçao, Netherlands Antilles, and collected from March 1999 to March 2000 were investigated by a range of molecular typing techniques. Data obtained by pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), automated ribotyping, and sequence analysis of the short variable region of the flagellin gene (flaA) were analyzed separately and in combination. Similar groupings were obtained by all methods, with the data obtained by MLST and AFLP analysis exhibiting the highest degree of congruency. MLST identified 29 sequence types, which were assigned to 10 major clonal complexes. PFGE, AFLP analysis, and ribotyping identified 10, 9, and 8 of these clonal groups, respectively; however, these three techniques permitted subdivision of the clonal groups into more different types. Members of seven clonal groups comprising 107 isolates were obtained from November 1999 to February 2000, and no distinguishing characteristics were identified for two GBS-associated strains. The sequence type 41 (ST-41), ST-508, and ST-657 clonal complexes and their corresponding AFLP types have been rare or absent in the Campylobacter data sets described to date. We conclude that several clonal complexes of C. jejuni are associated with human disease in Curaçao, and some of these have not been reported elsewhere. Furthermore, given the observation that C. jejuni-associated diseases appear to be more severe from November to February, it can be speculated that this may be due to the presence of virulent clones with a limited span of circulation.

Base Sequence↗
PubMed2004

Group A streptococci from a remote community have novel multilocus genotypes but share emm types and housekeeping alleles with isolates from worldwide sources.

Group A streptococci (GAS) cause several human diseases that differentially affect distinct host populations. Genotypes were defined by multilocus sequence typing and emm typing for 137 organisms collected from individuals in a remote aboriginal island community in tropical Australia and compared with >200 isolates obtained from sources elsewhere in the world. The majority of aboriginal-derived isolates shared emm types and housekeeping alleles with GAS isolates recovered from outside Australia, but these emm types and alleles were in novel combinations. There were many examples in which isolates from aboriginal and non-Australian subjects shared the same emm type, but for approximately 50% of emm types, the multilocus genotypes of isolates of the same emm type but from different regions were very different. A single emm type may typically define a single clone within the United States and on the remote island that is the focus of this study, but in many cases, these clones will be different, and this finding has implications for attempts to make global associations between emm types and certain disease manifestations.

Australia↗
PubMedDate not supplied

Molecular typing of pathogens.

Molecular typing and fingerprinting of bacterial pathogens has become a major part of epidemiology, disease monitoring, intervention and food safety research. This treatise will briefly address the aims of molecular typing and the criteria that are important to choose the optimal method of typing. However, since the method of choice will depend on a number of factors, including available facilities and research goals, no particular methods are recommended; instead, theoretical considerations are presented as a guide line for the best decision. Since molecular typing is examining the nature of the population genetics of a particular organism, it is important to understand how the degree of clonality of that particular organism will influence outcome and interpretation of results. The mechanisms that lead to non-clonality are briefly outlined. The problems with typing of clonal, non-clonal, and weakly clonal populations are briefly treated. The merits and myths of multilocus sequence typing (MLST) are discussed.

Bacteria↗
PubMed2026

Whole-Genome Sequencing of Feline Uropathogens Reveals Multidrug Resistance and Zoonotic Potential in Domestic Cats in Tunisia.

BACKGROUND: Urinary tract infections (UTIs) in cats are increasingly recognized as clinically relevant conditions frequently associated with multidrug-resistant (MDR) bacteria of potential zoonotic origin, yet genomic data on feline uropathogens remain scarce in Tunisia. METHODS: We used whole-genome sequencing to characterize seven bacterial isolates recovered from six cats with clinical signs of UTI: Mammaliicoccus lentus (n = 2), Staphylococcus schleiferi (n = 1), Mammaliicoccus sciuri (n = 1), Enterococcus faecalis (n = 1), Enterococcus casseliflavus (n = 1), and Klebsiella aerogenes (n = 1). RESULTS: Resistome analysis revealed determinants conferring resistance to β-lactams (blaZ, blaCMY-132), methicillin (mecC-type), macrolides (erm(43), ermB), tetracyclines (tet(M), tet(45), tetB), fosfomycins (fosI, fosB, fosA5), and aminoglycosides (aac(6'), aph(3')-IIIa, aph(6)-Id), alongside efflux pump genes (efrA, sepA, sdrM, oqxA, KpnE/F/G), vancomycin-operon genes (vanT, vanY, vanC, vanG), and biofilm/biocide-tolerance genes (salB, qacG). Notably, M. lentus S104 carried mecC-type elements, the first such report in Tunisia, while K. aerogenes displayed an extensive MDR profile, including blaCMY-132 and fosA5. Multilocus sequence typing/ribosomal multilocus sequence typing (MLST/rMLST) identified diverse lineages, including the internationally distributed E. faecalis ST19 and the rarely reported K. aerogenes ST242. Plasmids were absent in all isolates; a Tn916/1545-type transposon occurred in E. casseliflavus, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were unevenly distributed. CONCLUSIONS: These findings highlight companion animals as reservoirs of clinically important resistance genes, reinforcing the need for One Health AMR surveillance.

Animals↗
PubMed2006

AgdbNet - antigen sequence database software for bacterial typing.

BACKGROUND: Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. RESULTS: Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. CONCLUSION: The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated.

Algorithms↗
PubMed2005

Evidence for recombination in Candida glabrata.

Despite its clinical importance, little is known of the epidemiology and population structure of Candida glabrata. C. glabrata possesses a mating type system similar to that in Saccharomyces cerevisiae, however mating, meiosis and recombination have not been demonstrated. We performed multilocus sequence typing on a collection of 165 isolates to test for evidence of genetic recombination. A total of 3345 bp from six loci (FKS, LEU2, NMT1, TRP1, UGP1, and URA3) were sequenced for each isolate. The polymorphisms at these loci defined 34 sequence types. Significant evidence for a clonal population was revealed by the index of association and the number of phylogenetically compatible pairs of loci. However, 14 examples of phylogenetic incompatibility were also found. Thus we conclude that although C. glabrata has a predominantly clonal population structure, the multiple phylogenetic incompatibilities found strongly suggest that recombination occurred during the evolution of C. glabrata, and may infrequently still occur.

Candida glabrata↗
PubMed2006

Characterisation of Neisseria meningitidis C:2b:P1.2,5 isolates in Poland.

This study aimed to characterise Neisseria meningitidis C:2b:P1.2,5 isolates from Poland, which have now become predominant among serogroup C isolates in this country. Overall, 44 isolates (25 invasive and 19 from contact carriers) were typed by whole-cell ELISA and pulsed-field gel electrophoresis. Additionally, the invasive isolates were analysed by multilocus sequence typing, which revealed that they all belonged to the ST-8 complex/cluster A4. The emergence of this clone in other countries has resulted in mass immunisation campaigns and has been associated with a higher level of decreased susceptibility to penicillin; however the present study detected only one isolate that was penicillin-non-susceptible.

Female↗
PubMed2005

Dissemination of community-acquired methicillin-resistant Staphylococcus aureus clones in northern Norway: sequence types 8 and 80 predominate.

Increasing frequencies of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain isolation have been reported from many countries. The overall prevalence of MRSA in Norway is still very low. MRSA isolates (n = 67) detected between 1995 and 2003 in northern Norway were analyzed by pulsed-field gel electrophoresis, multilocus sequence typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Sixty-seven isolates were associated with 13 different sequence types. Two successful MRSA clones predominated. Sequence type 8 (ST8) (40%) and ST80 (19%) containing SCCmec type IV were detected in hospitals and communities in different geographic regions during a 7-year period. In general, there was a low level of antimicrobial resistance. Only 26% of the isolates were multiresistant. International epidemic clones were detected. The frequent findings of SCCmec type IV (91%) along with heterogeneous genetic backgrounds suggest a horizontal spread of SCCmec type IV among staphylococcal strains in parallel with the clonal spread of successful MRSA strains.

Community-Acquired Infections↗
PubMed2004

Sequence-based typing of flaB is a more stable screening tool than typing of flaA for monitoring of Campylobacter populations.

Different typing schemes for Campylobacter spp. were evaluated with 70 outbreak and sporadic isolates. The discriminatory indexes were 0.944 (by pulsed-field gel electrophoresis), 0.920 (by genotyping of the flagellin A gene), 0.902 (by genotyping of flaB), and 0.886 (by multilocus sequence typing). Cross-classification gave 94.77 or 95.82% (PFGE-flaA or PFGE-flaB) concordance. flaA was overdiscriminatory in three cases, most probably due to intragenomic recombination.

Animals↗
PubMed2003

Genetic diversity of Neisseria gonorrhoeae housekeeping genes.

Molecular typing of Neisseria gonorrhoeae strains is an important tool for epidemiological studies of gonococcal infection and transmission. The recently developed multilocus sequence typing (MLST) method is based on the genetic variation among housekeeping genes. As a preliminary investigation for the development of such a method, we characterized the genetic diversity at 18 gonococcal housekeeping gene loci. Approximately 17,500 nucleotides, spanning 18 loci, were sequenced from 24 isolates. Including strain FA 1090, which has been fully sequenced, and three unique glnA sequences from GenBank, the number of alleles identified for the 18 loci ranged from 2 to 18, with a mean of 8.3 alleles per locus. The majority of polymorphic sites were distributed randomly along the genes, consistent with evolution of DNA sequences by point mutation. In addition, several examples of clustered mutations and insertions or deletions were detected, which most likely occurred by recombinational events. While purifying selection is the dominant force driving the evolution of these housekeeping genes, positive selection also appeared to operate on the abcZ and gpdh loci. The 25 completely characterized strains each had a unique allelic profile with as few as three loci (pilA, abcZ, and pip or pgi2). Molecular typing based on the allelic profile of housekeeping genes resolved the isolates better than either porB nucleotide sequencing or typing of the opa gene. The allelic profiles for the pilA, abcZ, and serC loci of paired strains from 16 sexual contacts were identical. A potential MLST for N. gonorrhoeae, based on approximately 500- to 600-bp gene fragments of seven housekeeping gene loci, would include the pilA, abcZ, serC, glnA, gdh, gnd, and pip loci.

Base Sequence↗
PubMed2003

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children.

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.

Carrier State↗