Evidence of antimicrobial resistance-conferring genetic elements among pneumococci isolated prior to 1974.

BACKGROUND: Antimicrobial resistance among pneumococci has greatly increased over the past two to three decades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/or mef(A/E)) is generally conferred by acquisition of specific genes that are associated with mobile genetic elements, including those of the Tn916 and Tn5252 families. The first tetracycline-, chloramphenicol- and macrolide-resistant pneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown to harbour Tn916 and/or Tn5252 was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to 1974 were probed for the presence of tet(M), cat, erm(B), mef(A/E) and int (integrase) to indicate the presence of Tn916/Tn5252-like elements. RESULTS: Two Tn916-like, tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968. The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcal reference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third, novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1 contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novel lantibiotic synthesis/export-associated genes. CONCLUSIONS: These data confirm the existence of pneumococcal Tn916 elements in the first decade within which pneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates the dynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916 stability.

Pneumococcal capsular switching: a historical perspective.

BACKGROUND: Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. METHODS: Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events. RESULTS: We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥ 58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a. CONCLUSIONS: Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.

The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success.

BACKGROUND: Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species. RESULTS: We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described. CONCLUSIONS: These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.

Rapid pneumococcal evolution in response to clinical interventions.

Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain(23F)-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

Characterization and transfer studies of macrolide resistance genes in Streptococcus pneumoniae from Denmark.

Over the last decade, erythromycin resistance has been increasing in frequency in Streptococcus pneumoniae in Denmark. In the present study, 49 non-related erythromycin-resistant S. pneumoniae isolates from invasive sites and 20 isolates from non-invasive sites were collected; antimicrobial susceptibility was tested, and they were genotyped and serotyped. Gene transfer was studied for selected isolates. The frequency of erm(B) was significantly higher in non-invasive isolates compared to invasive isolates (p = 0.001). For the first time, mef(I) was detected in 1 isolate in Denmark. All tested mef(E) isolates had an identical mef(E) sequence, apart from 1 gene with a point mutation, and mef(E) was correlated to 7 different sero-types. The tested erm(B) sequences were 99.3% similar with 5 point mutations at different positions distributed among different serotypes, which did not cause a detectable influence on the protein. Transformation was detectable in 5 out of 13 isolates and transfer of erm(B), mef(I) and mef(E) was detected. To our knowledge, this is the first time mef(I) has been proved transformable. Gene transfer by conjugation was not detectable. Erythromycin resistance in pneumococcal isolates is likely to be caused primarily by horizontal spread of mef(E) and erm(B), as well as clonal spread of a serotype 14 strain carrying mef(A) primarily detected in invasive isolates.

Non-invasive erythromycin-resistant pneumococcal isolates are more often non-susceptible to more antimicrobial agents than invasive isolates.

Multiresistant Streptococcus pneumoniae infections are of great concern as treatment failures may occur with commonly used treatment regimens using beta-lactams and macrolides. The proportion of non-susceptible S. pneumoniae differs from country to country. In Denmark, the proportion of invasive penicillin- or erythromycin-non-susceptible isolates is still low. The aim of this study was to characterise and compare invasive and non-invasive penicillin-non-susceptible and erythromycin-resistant pneumococcal isolates from the same geographic area and the same time period with respect to serotype and antibiotic susceptibility profile. We aimed to identify which serotypes were multiresistant among Danish isolates and to confirm or reject whether there was a difference in serotype distribution and resistance profiles between invasive and non-invasive isolates. We observed that non-invasive penicillin-non-susceptible pneumococci had higher serotype diversity than invasive isolates. This was not the case for erythromycin-resistant pneumococci. The dominant serotypes among non-susceptible invasive isolates were serotypes 9V and 14, whereas the dominant serotypes among non-susceptible non-invasive isolates were serotypes 19F, 14, 9V, 6B and non-typeable (NT). Non-invasive isolates were also more likely to be resistant to three or more antimicrobial agents than invasive isolates, however isolates being multiresistant were often co-resistant to the same antimicrobial agents.

Transcriptional regulation of pWW0 transfer genes in Pseudomonas putida KT2440.

The conjugative IncP-9 plasmid pWW0 (TOL) carries transfer genes, many of whose functions can be predicted from sequence similarities to the well-studied IncW and IncP-1 plasmids, and that are clustered with the replication and maintenance genes of the plasmid core. In this study we show that the IncP-9 transfer genes are transcribed from at least three promoter regions. The promoters for traA and traD act divergently from the region found to encode the origin of transfer, oriT. These promoters regulate expression of traA, B, and perhaps traC in one direction and traD in the other, all of whose gene products are predicted to be involved in relaxasome formation and DNA processing during transfer, and they are repressed by TraA. The third promoter region, upstream of mpfR, is responsible for transcription of mpfR and mpfA to mpfJ, encoding proteins involved in mating pair formation. Transcription from this region is negatively autoregulated by MpfR. Thus the pWW0 transfer genes, like those of the IncP-1 plasmids, are expressed at all times, but kept in control by a negative feed back loop to limit the metabolic burden on the host. Although many of the related mating pair formation systems are, as in pWW0, transcribed divergently from an operon for replication and/or stable inheritance functions, MpfR is not related to the known regulatory proteins of these other transfer systems outside those of the IncP-9 family and indeed the regulators tend to be specific for each plasmid family. This suggests that the general pattern of genetic organisation exhibited by these systems has arisen a number of times independently and must therefore be highly favourable to plasmid survival and spread.