Geographical migration and fitness dynamics of Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location1,2. The extent and mechanisms of spread and vaccine-driven changes in fitness and antimicrobial resistance remain largely unquantified. Here using geolocated genome sequences from South Africa (n = 6,910, collected from 2000 to 2014), we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately, we estimated the population-level changes in fitness of strains that are included (vaccine type (VT)) and not included (non-vaccine type (NVT)) in pneumococcal conjugate vaccines, first implemented in South Africa in 2009. Differences in strain fitness between those that are and are not resistant to penicillin were also evaluated. We found that pneumococci only become homogenously mixed across South Africa after 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Furthermore, in the years following vaccine implementation, the relative fitness of NVT compared with VT strains increased (relative risk of 1.68; 95% confidence interval of 1.59-1.77), with an increasing proportion of these NVT strains becoming resistant to penicillin. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in antimicrobial resistance may be transient.

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Streptococcus suis serotype 9 in Italy: genomic insights into high-risk clones with emerging resistance to penicillin.

BACKGROUND: Streptococcus suis is an important pig pathogen and an emerging zoonotic agent. In a previous study, we described a high proportion of penicillin-resistant serotype 9 S. suis (SS9) isolates on pig farms in Italy. OBJECTIVES: We hypothesized that resistance to penicillin emerged in some SS9 lineages characterized by substitutions at the PBPs, contributing to the successful spread of these lineages in the last 20 years. METHODS: Sixty-six SS9 isolates from cases of streptococcosis in pigs were investigated for susceptibility to penicillin, ceftiofur and ampicillin. The isolates were characterized for ST, virulence profile, and antimicrobial resistance genes through WGS. Multiple linear regression models were employed to investigate the associations between STs, year of isolation, substitutions at the PBPs and an increase in MIC values to ß-lactams. RESULTS: MIC values to penicillin increased by 4% each year in the study period. Higher MIC values for penicillin were also positively associated with ST123, ST1540 and ST1953 compared with ST16. The PBP sequences presented a mosaic organization of blocks. Within the same ST, substitutions at the PBPs were generally more frequent in recent isolates. Resistance to penicillin was driven by substitutions at PBP2b, including K479T, D512E and K513E, and PBP2x, including T551S, while reduced susceptibility to ceftiofur and ampicillin were largely dependent on substitutions at PBP2x. CONCLUSIONS: Here, we identify the STs and substitutions at the PBPs responsible for increased resistance of SS9 to penicillin on Italian pig farms. Our data highlight the need for monitoring the evolution of S. suis in the coming years.

The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci.

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to ß-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965-2020.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The PenNS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

Penicillin resistance in bovine Staphylococcus aureus: Genomic evaluation of the discrepancy between phenotypic and molecular test methods.

Staphylococcus aureus is a major pathogen in humans and animals. In cattle, it is one of the most important agents of mastitis, causing serious costs in the dairy industry. Early diagnosis and adequate therapy are therefore 2 key factors to deal with the problems caused by this bacterium, and benzylpenicillin (penicillin) is usually the first choice to treat these infections. Unfortunately, penicillin resistance testing in bovine S. aureus strains shows discrepant results depending on the test used; consequently, the best method for assessing penicillin resistance is still unknown. The aim of this study was therefore to find a method that assesses penicillin resistance in S. aureus and to elucidate the mechanisms leading to the observed discrepancies. A total of 146 methicillin-sensitive S. aureus strains isolated from bovine mastitis were tested for penicillin resistance using a broth microdilution [minimum inhibitory concentration (MIC)] and 2 different disk diffusion protocols. Furthermore, the strains were analyzed for the presence of the bla operon genes (blaI, blaR1, blaZ) by PCR, and a subset of 45 strains was also subjected to whole genome sequencing (WGS). Discrepant results were obtained when penicillin resistance of bovine S. aureus was evaluated by disk diffusion, MIC, and PCR methods. The discrepancies, however, could be fully explained by WGS analysis. In fact, it turned out that penicillin resistance is highly dependent on the completeness of the bla operon promotor: when the bla operon was complete based on WGS analysis, all strains showed MIC ≥1 µg/mL, whereas when the bla operon was mutated (31-nucleotide deletion), they were penicillin sensitive except in those strains where an additional, bla operon-independent resistance mechanism was observed. Further, WGS analyses showed that penicillin resistance is truly assessed by the MIC assay. In contrast, caution is required when interpreting disk diffusion and PCR results.

Serogroup Y Clonal Complex 23 Meningococcus in China Acquiring Penicillin Resistance from Commensal Neisseria lactamica Species.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (NmY) is rare in China; recently, an invasive NmY isolate, Nm512, was discovered in Shanghai with decreased susceptibility to penicillin (PenNS). Here, we investigated the epidemiology of NmY isolates in Shanghai and explored the potential commensal Neisseria lactamica donor of the PenNS NmY isolate. A total of 491 N. meningitidis and 724 commensal Neisseria spp. isolates were collected. Eleven NmY isolates were discovered from IMD (n = 1) and carriers (n = 10), including two PenNS isolates with five-key-mutation-harboring (F504L-A510V-I515V-H541N-I566V) penA genes. Five of the eight ST-175 complex (CC175) isolates had a genotype [Y:P1.5-1,2-2:F5-8:ST-175(CC175)] identical to that of the predominant invasive clone found in South Africa. Only one invasive NmY CC23 isolate (Nm512) was discovered; this isolate carried a novel PenNSpenA832 allele, which was identified in commensal N. lactamica isolates locally. Recombination analysis and transformation of the penA allele highlighted that N. meningitidis Nm512 may acquire resistance from its commensal donor; this was supported by the similar distribution of transformation-required DNA uptake sequence variants and the highly cognate receptor ComP between N. meningitidis and N. lactamica. In 2,309 NmY CC23 genomes from the PubMLST database, isolates with key-mutation-harboring penA genes comprised 12% and have been increasing since the 1990s, accompanied by recruitment of the blaROB-1 and/or quinolone resistance allele. Moreover, penA22 was predominant among genomes without key mutations in penA. These results strongly suggest that Nm512 is a descendant of the penA22-harboring CC23 isolate from Europe and acquired its penicillin resistance locally from commensal N. lactamica species by natural transformation.

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae.

PURPOSE: In Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. MATERIALS AND METHODS: Pneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to ß-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. RESULTS: In PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. CONCLUSIONS: NVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.

Smart pH-Regulated Switchable Nanoprobes for Photoelectrochemical Multiplex Detection of Antibiotic Resistance Genes.

Antibiotic resistance, encoded via particular genes, has become a major global health threat and substantial burden on healthcare. Hence, the facile, low-cost, and precise detection of antibiotic resistance genes (ARGs) is crucial in the realm of human health and safety, especially multiplex sensing assays. Here, a smart pH-regulated switchable photoelectrochemical (PEC) bioassay has been created for ultrasensitive detection of two typical subtypes of penicillin resistance genes bla-CTX-M-1 (target 1, labeled as TDNA1) and bla-TEM (target 2, labeled as TDNA2), whereby pH-responsive antimony tartrate (SbT) complex-grafted silica nanospheres are ingeniously adopted as signal DNA1 tags (labeled as SDNA1-SbT@SiO2NSs). The operations of the PEC bioassay depend on the switchable dissociation of the pH-responsive SDNA1-SbT@SiO2NSs complex under the external pH stimuli, thus initiating the pH-regulated release of ions pre-embedded in sandwich-type DNA nanoassemblies. At acidic conditions, the dissociation of SDNA1 tags (ON state) triggers the release of the embedded SbO+. Under alkaline conditions, the dissociation of SDNA1 tags is inhibited (OFF state). The detection of TDNA2 was achieved via DNA hybridization-triggered metal ion release. The unwinding of the introduced hairpin T-Hg2+-T fragment, hybridized with the second anchored signal DNA (SDNA2), ignites the release of Hg2+. The released SbO+ or Hg2+ ions would trigger the formation of Sb2S3/ZnS or HgS/ZnS heterostructure through ion-exchange with the photosensitive ZnS layer, giving rise to the amplified photocurrents and eventually realizing the ultrasensitive detection of penicillin resistance genes subtypes, bla-CTX-M-1 and bla-TEM. The as-fabricated pH-regulated PEC bioassay, smartly integrating the pH-responsive intelligent unit as SDNA tags, pH-regulated release of embedded ions, and the subsequent ion-exchange-based signal amplification strategy, exhibits high sensitivity, specificity, low-cost, and ease of use for multiplex detection of ARGs. It can be successfully used for measuring bla-CTX-M-1 and bla-TEM in real E. coli plasmids, demonstrating great promise for developing a new class of genetic point-of-care devices.

Performance of penicillinase detection tests in Staphylococcus epidermidis: comparison of different phenotypic methods.

BACKGROUND: Staphylococcus epidermidis is the leading coagulase negative staphylococci (CoNS) species associated with healthcare associated infections. In order to de-escalate antimicrobial therapy, isolates of S. epidermidis lacking the blaZ gene should be eligible for targeted antimicrobial therapy. However, testing the susceptibility of coagulase negative staphylococci (CoNS) to penicillin G is no longer recommended by EUCAST, given the low performances for penicillinase detection in CoNS. The objective of this work was to determine a phenotypic method with high performance for detecting penicillinase production in S. epidermidis. RESULTS: Four techniques for the detection of penicillinase production (disk diffusion, zone edge test, nitrocefin test, Minimal Inhibitory Concentration (MIC) by automated system Vitek2®) were evaluated on 182 S. epidermidis isolates, using identification of blaZ gene by PCR as the reference method. The performance of the methods for penicillinase detection was compared by the sensitivity, the specificity, the negative predictive value and the positive predictive value, and with Cohen's kappa statistical test. Among the 182 S. epidermidis included in this study, 55 carried the blaZ gene. The nitrocefin test, characterized by a poor sensitivity (91%), was therefore excluded from S. epidermidis penicillinase detection. The algorithm proposed here for the penicillinase detection in S. epidermidis involved two common antimicrobial susceptibility techniques: disk diffusion method and MIC by Vitek2® system. Disk diffusion method, interpreted with a 26 mm breakpoint for penicillin G, was associated with a high sensitivity (98%) and specificity (100%). This method was completed with zone edge test for S. epidermidis with penicillin G diameter from 26 to 35 mm (sensitivity of 98%). The Vitek2® system is associated with a low sensitivity (93%) and a high specificity (99%) This low sensitivity is associated with false negative results, in isolates with 0.12 mg/L Penicillin G MIC values and blaZ positive. Thus for penicillin G MIC of 0.06 mg/L or 0.12 mg/L, a second step with disc diffusion method is suggested. CONCLUSIONS: According to our results, the strategy proposed here allows the interpretation of penicillin G susceptibility in S. epidermidis isolates, with an efficient detection of penicillin G resistance.

Isolation and characterization of amoxicillin-resistant bacteria and amoxicillin-induced alteration in its protein profiling and RNA yield.

Amoxicillin-resistant bacteria were isolated using selective enrichment procedure. The morphological, biochemical and molecular characterization based on 16S rRNA gene sequencing and phylogenetic analysis of the bacterial strain WA5 confirmed that the strain belongs to the genus Stenotrophomonas. The bacteria were named as Stenotrophomonas sp. strain WA5 (MK110499). Substantial growth was seen in M9 minimal media supplemented with 5 mg L-1 of amoxicillin as a sole source of carbon and energy. RNA yield was also observed to be decreased in the presence of amoxicillin. Amoxicillin (5 mg L-1)-induced alteration is seen on bacterial protein profile and unique polypeptide bands were seen to be induced in the presence of amoxicillin, the bands were subjected to trypsin digestion, and LC-MS/MS analysis showed that the bands belong to the family of DNA-dependent RNA polymerase subunit ß (rpoC). Plasmid DNA isolation indicated the presence of antibiotic-resistant genes being harboured by the plasmid.

Escherichia coli as a carrier of tetracyclines and penicillins resistance in wild pheasant (Phasianus colchicus).

Wild animals like pheasant seem to be a good source of information about human activities. Therefore, the wild pheasants and relative stable appendix microcenosis were selected for antibiotic resistance testing. Penicillin resistance by MALDI-TOF Mass Spectrometry and tetracyclines resistance by genetic methods using specific primers were tested. Differences between tetracycline and penicillin resistance were detected. Results showed high prevalence of resistant Escherichia coli isolated from wild pheasant appendix. E. coli isolated from wild pheasant appendix carried plasmids for penicillins and tetracyclines resistance where they were responsible for enzymatic degradation of penicillin and carried genes for regulating efflux pumps for tetracyclines. Results showed that tetracyclines and penicillins resistance is widespread between wild pheasants with a carrier as Escherichia coli isolated from relative stable microcenosis of appendix.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.

Staphylococcus aureus is one of the leading causes of food-borne illness worldwide. Raw milk and dairy products are often contaminated with enterotoxigenic strains of this bacterium. Some of these strains carry antimicrobial resistance, leading to a potential risk for consumers. The aim of this study was to characterize S. aureus strains circulating in raw milk and traditional dairy products for carriage of staphylococcal enterotoxin (se) genes and antimicrobial resistance. Overall, 62 out of 270 samples (23%) were contaminated with S. aureus, and 69 S. aureus strains were identified. We studied the enterotoxin genes using 2 multiplex PCR targeting 11 se genes. Seventeen (24.6%) isolates carried one or more genes encoding for staphylococcal enterotoxins. The most commonly detected se genes were seb and sep, followed by seh, sea, and see. Using the disk diffusion method, we found that resistance to penicillin G and tetracycline was the most common. Eleven isolates of methicillin-resistant S. aureus (MRSA) carried the mecA gene. All MRSA isolates belonged to the same spa type (t024) and sequence type (ST8), and carried the seb and sep enterotoxin genes. However, none of them carried the Panton Valentine leukocidin gene (lukF/S-PV). The presence of enterotoxigenic S. aureus strains, including MRSA, in raw milk and dairy products, raises a serious public health concern, because these strains may cause food poisoning outbreaks, be disseminated to the population, or both.

Effects of Pneumococcal Conjugate Vaccine on Genotypic Penicillin Resistance and Serotype Changes, Japan, 2010-2017.

To clarify year-to-year changes in capsular serotypes, resistance genotypes, and multilocus sequence types of Streptococcus pneumoniae, we compared isolates collected from patients with invasive pneumococcal disease before and after introductions of 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PVC13, respectively). From April 2010 through March 2017, we collected 2,856 isolates from children and adults throughout Japan. Proportions of PCV13 serotypes among children decreased from 89.0% in fiscal year 2010 to 12.1% in fiscal year 2016 and among adults from 74.1% to 36.2%. Although nonvaccine serotypes increased after introduction of PCV13, genotypic penicillin resistance decreased from 54.3% in 2010 to 11.2% in 2016 among children and from 32.4% to 15.5% among adults. However, genotypic penicillin resistance emerged in 9 nonvaccine serotypes, but not 15A and 35B. Multilocus sequence typing suggested that resistant strains among nonvaccine serotypes may have evolved from clonal complexes 156 and 81. A more broadly effective vaccine is needed.

Molecular Characterization of Invasive Isolates of Neisseria meningitidis in Casablanca, Morocco.

Meningococcal epidemiology may change unpredictably, and typing of Neisseria meningitidis isolates is crucial for the surveillance of invasive meningococcal disease (IMD). Few data are available regarding the meningococcal epidemiology in countries of North Africa. We aimed to explore invasive meningococcal isolates from the Casablanca region in Morocco. We used whole-genome sequencing (WGS) to characterize 105 isolates from this region during the period of 2011 to 2016. Our data showed that the majority (n = 100) of the isolates belonged to serogroup B. Genotyping indicated that most of the isolates (n = 62) belonged to sequence type 33 of clonal complex 32. The isolates also showed the same PorA and FetA markers and clustered together on the basis of WGS phylogenetic analysis; they seemed to correspond to an expansion of local isolates in the Casablanca region, as reported for similar isolates in several other countries. These data suggest that serogroup B isolates may predominate in Morocco, which may have an important impact in the design of vaccination strategies.

Molecular Antimicrobial Resistance of Neisseria gonorrhoeae in a Moroccan Area.

Objectives: To identify the prevalence and the types of Neisseria gonorrhoeae (NG) resistance plasmids-mediated penicillin (PPNG) and tetracycline (TRNG), the ciprofloxacin resistance (CRNG), and related risk factors of each types of resistance. Methods: The beta-lactamase-producing plasmid types (Africa, Asia, and Toronto), tetM tetracycline resistance plasmid types (America and Dutch), and the determination of the Ser-91 mutation of GyrA were detected by specifics PCRs on 149 diagnosed NG positives samples followed by Hinf1 digestion for tetM and gyrA mutation. Results: 135 (90.1%) samples showed a profile of molecular resistance to at least one antibiotic with predominance of ciprofloxacin resistance. In fact, 36 (24.2%) and 69 (46.3%) cases harbored PPNG and TRNG, respectively, and 116 (77.9%) cases showed the mutation Ser-91 of GyrA (CRNG). From a total of 36 PPNG isolates, the Toronto, Asian, and Toronto/Asian types were detected in 13 (36.1%), 10 (27.8%), and 13 (36.1%) cases, respectively, whereas the African type was not detected. In addition, the American type of TRNG was detected in 92.8% (64/69) of cases, while the Dutch type was detected in 7.2% (5/69) of cases. The association of demographics and clinical variables with NG resistance to ciprofloxacin, penicillin, and tetracycline was studied and the risk factors have been determined. Conclusion: Resistance to penicillin, tetracycline, and ciprofloxacin among NG samples positives remained at high levels in Morocco as determined by molecular profile. So, the use of molecular tools for NG antimicrobial resistance detection can help in the management and spread limitation of this infection.

Clonal Expansion of New Penicillin-Resistant Clade of Neisseria meningitidis Serogroup W Clonal Complex 11, Australia.

In Western Australia, Neisseria meningitidis serogroup W clonal complex 11 became the predominant cause of invasive meningococcal disease in 2016. We used core-genome analysis to show emergence of a penicillin-resistant clade that had the penA_253 allele. This new penicillin-resistant clade might affect treatment regimens for this disease.

Genome Evolution to Penicillin Resistance in Serotype 3 Streptococcus pneumoniae by Capsular Switching.

Streptococcus pneumoniae isolates of serotype 3 were collected from cases of invasive pneumococcal disease (n = 124) throughout Japan between April 2010 and March 2013. A penicillin-resistant S. pneumoniae (PRSP) isolate from an adult patient, strain KK0981 of serotype 3, was identified among these strains. Whole-genome analysis characterized this PRSP as a recombinant strain derived from PRSP of serotype 23F with the cps locus (20.3 kb) replaced by that of a penicillin-susceptible strain of serotype 3.

Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci.

The identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis, and S. infantis and S. oralis sequences were found in S. pneumoniae from Japan. PBP2x genes of the family of mosaic genes related to pbp2x in the S. pneumoniae clone Spain23F-1 were observed in S. oralis and S. infantis as well, confirming its global distribution. Thirty-eight sites were altered within the PBP2x transpeptidase domains of penicillin-resistant strains, excluding another 37 sites present in the reference genes of sensitive strains. Specific mutational patterns were detected depending on the parental sequence blocks, in agreement with distinct mutational pathways during the development of beta-lactam resistance. The majority of the mutations clustered around the active site, whereas others are likely to affect stability or interactions with the C-terminal domain or partner proteins.

Diversity of Mosaic pbp2x Families in Penicillin-Resistant Streptococcus pneumoniae from Iran and Romania.

Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for ß-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.