Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania.

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

New Resistance Mutations Linked to Decreased Susceptibility to Solithromycin in Streptococcus pneumoniae Revealed by Chemogenomic Screens.

Two strains of Streptococcus pneumoniae, one expressing the methyltransferase Erm(B) and the other negative for erm(B), were selected for solithromycin resistance in vitro either with direct drug selection or with chemical mutagenesis followed by drug selection. We obtained a series of mutants that we characterized by next-generation sequencing. We found mutations in various ribosomal proteins (L3, L4, L22, L32, and S4) and in the 23S rRNA. We also found mutations in subunits of the phosphate transporter, in the DEAD box helicase CshB, and in the erm(B)L leader peptide. All mutations were shown to decrease solithromycin susceptibility when transformed into sensitive isolates. Some of the genes derived from our in vitro screens were found to be mutated also in clinical isolates with decreased susceptibility to solithromycin. While many mutations were in coding sequences, some were found in regulatory regions. These included novel phenotypic mutations in the intergenic regions of the macrolide resistance locus mef(E)/mel and in the vicinity of the ribosome binding site of erm(B). Our screens highlighted that macrolide-resistant S. pneumoniae can easily acquire resistance to solithromycin, and they revealed many new phenotypic mutations.

Exploiting antimicrobial resistance: Better knowledge of resistance mechanisms can inform the search for and development of new antibiotics.

Antibiotic resistance is a grave threat for public health. Understanding the mechanisms of resistance could lead to new drugs and therapeutic strategies against resistant pathogens.

Comparison of the Wechsler Preschool and Primary Scale of Intelligence-Third Edition and the Leiter-R Intellectual Assessments for Clinic-Referred Children.

A review of clinical records was conducted for children with developmental, emotional, and behavioral difficulties who were assessed with both the Wechsler preschool and primary scale of intelligence-third edition (WPPSI-IIICDN; Wechsler, 2004) and the Leiter international performance scale-revised (Leiter-R; Roid & Miller, 1997) within the same psychological evaluation. Forty children, ages 3-7, were included in this study. Pearson correlations showed that the IQ scores of the two instruments are strongly related (r > .70; p < .001). However, paired t-tests showed that overall Leiter-R scores (M = 99.03) were significantly higher than WPPSI-IIICDN scores (PIQ; M = 82.28, FSIQ; M = 75.24) (p < .001). The discrepancies between the instrument's scores were clinically important as the use of only one of the two instruments could result in misclassification of child intellectual ability. These results should prompt professionals working with this clinical population to be cautious when using results from a single instrument in a child's intellectual evaluation.

Mutations in an Aquaglyceroporin as a Proven Marker of Antimony Clinical Resistance in the Parasite Leishmania donovani.

BACKGROUND: Antimonial drugs have long been the mainstay to treat visceral leishmaniasis. Their use has been discontinued in the Indian subcontinent because of drug resistance, but they are still clinically useful elsewhere. The goal of this study was to find markers of antimony resistance in Leishmania donovani clinical isolates and validate experimentally their role in resistance. METHODS: The genomes of sensitive and antimony-resistant clinical isolates were sequenced. The role of a specific gene in contributing to resistance was studied by CRISPR-Cas9-mediated gene editing and intracellular drug sensitivity assays. RESULTS: Both gene copy number variations and single nucleotide variants were associated with antimony resistance. A homozygous insertion of 2 nucleotides was found in the gene coding for the aquaglyceroporin AQP1 in both resistant isolates. Restoring the wild-type AQP1 open reading frame re-sensitized the 2 independent resistant isolates to antimonials. Alternatively, editing the genome of a sensitive isolate by incorporating the 2-nucleotide insertion in its AQP1 gene led to antimony-resistant parasites. CONCLUSIONS: Through genomic analysis and CRISPR-Cas9-mediated genome editing we have proven the role of the AQP1 mutations in antimony clinical resistance in L. donovani.

Gain- and Loss-of-Function Screens Coupled to Next-Generation Sequencing for Antibiotic Mode of Action and Resistance Studies in Streptococcus pneumoniae.

Two whole-genome screening approaches are described for studying the mode of action and the mechanisms of resistance to trimethoprim (TMP) in the Gram-positive Streptococcus pneumoniae The gain-of-function approach (Int-Seq) relies on a genomic library of DNA fragments integrated into a fucose-inducible cassette. The second approach, leading to both gain- and loss-of-function mutation, is based on chemical mutagenesis coupled to next-generation sequencing (Mut-Seq). Both approaches pointed at the drug target dihydrofolate reductase (DHFR) as a major resistance mechanism to TMP. Resistance was achieved by dhfr overexpression either through the addition of fucose (Int-Seq) or by mutations upstream of the gene (Mut-Seq). Three types of mutations increased expression by disrupting a predicted Rho-independent terminator upstream of dhfr Known and novel DHFR mutations were also detected by Mut-Seq, and these were functionally validated for TMP resistance. The two approaches also suggested that an increase in the metabolic flux from purine synthesis to GTP and then to folate can modulate the susceptibility to TMP. Finally, we provide evidence for a novel role of the ABC transporter PatAB in TMP susceptibility. Our genomic screens highlighted novel aspects on the mode of action and mechanisms of resistance to antibiotics.

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.

Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.

Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism.

The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.

Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.

Engagement of the Aryl Hydrocarbon Receptor in Mycobacterium tuberculosis-Infected Macrophages Has Pleiotropic Effects on Innate Immune Signaling.

Understanding the mechanisms of host macrophage responses to Mycobacterium tuberculosis is essential for uncovering potential avenues of intervention to boost host resistance to infection. Macrophage transcriptome profiling revealed that M. tuberculosis infection strongly induced the expression of several enzymes controlling tryptophan catabolism. These included IDO1 and tryptophan 2,3-dioxygenase, which catalyze the rate-limiting step in the kynurenine pathway, producing ligands for the aryl hydrocarbon receptor (AHR). The AHR and heterodimeric partners AHR nuclear translocator and RELB are robustly expressed, and AHR and RELB levels increased further during infection. Infection enhanced AHR/AHR nuclear translocator and AHR/RELB DNA binding and stimulated the expression of AHR target genes, including that encoding the inflammatory cytokine IL-1ß. AHR target gene expression was further enhanced by exogenous kynurenine, and exogenous tryptophan, kynurenine, or synthetic agonist indirubin reduced mycobacterial viability. Comparative expression profiling revealed that AHR ablation diminished the expression of numerous genes implicated in innate immune responses, including several cytokines. Notably, AHR depletion reduced the expression of IL23A and IL12B transcripts, which encode subunits of IL-23, a macrophage cytokine that stimulates production of IL-22 by innate lymphoid cells. AHR directly induced IL23A transcription in human and mouse macrophages through near-upstream enhancer regions. Taken together, these findings show that AHR signaling is strongly engaged in M. tuberculosis-infected macrophages and has widespread effects on innate immune responses. Moreover, they reveal a cascade of AHR-driven innate immune signaling, because IL-1ß and IL-23 stimulate T cell subsets producing IL-22, another direct target of AHR transactivation.

Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum.

To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival.

Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

Deep-sequencing revealing mutation dynamics in the miltefosine transporter gene in Leishmania infantum selected for miltefosine resistance.

Miltefosine is the first oral drug used in chemotherapy against leishmaniasis. In vitro studies found that resistance to miltefosine in Leishmania is often associated with the acquisition of point mutations in the miltefosine transporter, leading to a decrease in drug uptake. In this study, the dynamics of mutations upon miltefosine selection was studied by deep-sequencing of the miltefosine transporter gene. Deep-sequencing data revealed that no mutation was detected in the miltefosine transporter at sub-inhibitory concentrations of miltefosine. We show that the prevalence of mutated alleles was increasing when the drug pressure heightened, that more mutations were observed in highly resistant mutants, and that most mutations remained when parasites were cultured for a few passages in the absence of miltefosine.

A genomic approach to understand interactions between Streptococcus pneumoniae and its bacteriophages.

BACKGROUND: Bacteriophage replication depends on bacterial proteins and inactivation of genes coding for such host factors should interfere with phage infection. To gain further insights into the interactions between S. pneumoniae and its pneumophages, we characterized S. pneumoniae mutants selected for resistance to the virulent phages SOCP or Dp-1. RESULTS: S. pneumoniae R6-SOCP(R) and R6-DP1(R) were highly resistant to the phage used for their selection and no cross-resistance between the two phages was detected. Adsorption of SOCP to R6-SOCP(R) was partly reduced whereas no difference in Dp-1 adsorption was noted on R6-DP1(R). The replication of SOCP was completely inhibited in R6-SOCP(R) while Dp-1 was severely impaired in R6-DP1(R). Genome sequencing identified 8 and 2 genes mutated in R6-SOCP(R) and R6-DP1(R), respectively. Resistance reconstruction in phage-sensitive S. pneumoniae confirmed that mutations in a GntR-type regulator, in a glycerophosphoryl phosphodiesterase and in a Mur ligase were responsible for resistance to SOCP. The three mutations were additive to increase resistance to SOCP. In contrast, resistance to Dp-1 in R6-DP1(R) resulted from mutations in a unique gene coding for a type IV restriction endonuclease. CONCLUSION: The characterization of mutations conferring resistance to pneumophages highlighted that diverse host genes are involved in the replication of phages from different families.

Differences in antibiotic-induced oxidative stress responses between laboratory and clinical isolates of Streptococcus pneumoniae.

Oxidants were shown to contribute to the lethality of bactericidal antibiotics in different bacterial species, including the laboratory strain Streptococcus pneumoniae R6. Resistance to penicillin among S. pneumoniae R6 mutants was further shown to protect against the induction of oxidants upon exposure to unrelated bactericidal compounds. In the work described here, we expanded on these results by studying the accumulation of reactive oxygen species in the context of antibiotic sensitivity and resistance by including S. pneumoniae clinical isolates. In S. pneumoniae R6, penicillin, ciprofloxacin, and kanamycin but not the bacteriostatic linezolid, erythromycin, or tetracycline induced the accumulation of reactive oxygen species. For the three bactericidal compounds, resistance to a single molecule prevented the accumulation of oxidants upon exposure to unrelated bactericidal antibiotics, and this was accompanied by a reduced lethality. This phenomenon does not involve target site mutations but most likely implicates additional mutations occurring early during the selection of resistance to increase survival while more efficient resistance mechanisms are being selected or acquired. Bactericidal antibiotics also induced oxidants in sensitive S. pneumoniae clinical isolates. The importance of oxidants in the lethality of bactericidal antibiotics was less clear than for S. pneumoniae R6, however, since ciprofloxacin induced oxidants even in ciprofloxacin-resistant S. pneumoniae clinical isolates. Our results provide a clear example of the complex nature of the mode of action of antibiotics. The adaptive approach to oxidative stress of S. pneumoniae is peculiar, and a better understanding of the mechanism implicated in response to oxidative injury should also help clarify the role of oxidants induced by antibiotics.

Induced tigecycline resistance in Streptococcus pneumoniae mutants reveals mutations in ribosomal proteins and rRNA.

OBJECTIVES: Tigecycline is a broad-spectrum antibiotic acting at the level of the 30S ribosomal subunit to inhibit translation. While Streptococcus pneumoniae remains susceptible to tigecycline, resistance is beginning to emerge in some species and mainly involves efflux or mutations in ribosome constituents. We describe here the characterization of S. pneumoniae mutants selected for resistance to tigecycline. METHODS: Molecular determinants of resistance to tigecycline in S. pneumoniae were studied through WGS of two series of mutants made resistant to tigecycline in vitro in a stepwise fashion and by reconstructing tigecycline resistance using DNA transformation. RESULTS: The tigecycline-resistant S. pneumoniae M1TGC-6 and M2TGC-6 mutants were cross-resistant to tetracycline and minocycline. A role in tigecycline resistance could be attributed to 4 of the 12 genes that were mutated in both mutants. Mutations in ribosomal proteins S10 and S3, acquired early and late during selection, respectively, were implicated in resistance in both mutants. Similarly, mutations were detected in the four alleles of the 16S ribosomal RNA at sites involved in tigecycline binding and the number of mutated alleles correlated with the level of resistance. Finally, the gene spr1784 encodes an RsmD-like 16S rRNA methyltransferase for which inactivating mutations selected in the S. pneumoniae tigecycline-resistant mutants were found to decrease susceptibility to tigecycline. CONCLUSIONS: This first report about tigecycline resistance mechanisms in S. pneumoniae revealed that, in contrast to Gram-negative species, for which efflux appears central for tigecycline resistance, resistance in the pneumococcus occurs through mutations related to ribosomes.

Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing.

OBJECTIVES: The objective of this study was to characterize chromosomal mutations associated with resistance to tetracycline in Streptococcus pneumoniae. METHODS: Chronological appearance of mutations in two S. pneumoniae R6 mutants (R6M1TC-5 and R6M2TC-4) selected for resistance to tetracycline was determined by next-generation sequencing. A role for the mutations identified was confirmed by reconstructing resistance to tetracycline in a S. pneumoniae R6 WT background. RNA sequencing was performed on R6M1TC-5 and R6M2TC-4 and the relative expression of genes was reported according to R6. Differentially expressed genes were classified according to their ontology. RESULTS: WGS of R6M1TC-5 and R6M2TC-4 revealed mutations in the gene rpsJ coding for the ribosomal protein S10 and in the promoter region and coding sequences of the ABC genes patA and patB. These cells were cross-resistant to ciprofloxacin. Resistance reconstruction confirmed a role in resistance for the mutations in rpsJ and patA. Overexpression of the ABC transporter PatA/PatB or mutations in the coding sequence of patA contributed to resistance to tetracycline, ciprofloxacin and ethidium bromide, and was associated with a decreased accumulation of [(3)H]tetracycline. Comparative transcriptome profiling of the resistant mutants further revealed that, in addition to the overexpression of patA and patB, several genes of the thiamine biosynthesis and salvage pathway were increased in the two mutants, but also in clinical isolates resistant to tetracycline. This overexpression most likely contributes to the tetracycline resistance phenotype. CONCLUSIONS: The combination of genomic and transcriptomic analysis coupled to functional studies has allowed the discovery of novel tetracycline resistance mutations in S. pneumoniae.

Telomeric gene deletion and intrachromosomal amplification in antimony-resistant Leishmania.

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next-generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony-resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate-dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes.

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles.

Genomic analyses of DNA transformation and penicillin resistance in Streptococcus pneumoniae clinical isolates.

Alterations in penicillin-binding proteins, the target enzymes for ß-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.