Penicillin-binding Protein Genes Research Articles

Mapping-free variant calling using haplotype reconstruction from k-mer frequencies.

MotivationThe standard protocol for detecting variation in DNA is to map millions of short sequence reads to a known reference and find loci that differ. While this approach works well, it cannot be applied where the sample contains dense variants or is too distant from known references. De novo assembly or hybrid methods can recover genomic variation, but the cost of computation is often much higher. We developed a novel k-mer algorithm and software implementation, Kestrel, capable of characterizing densely packed SNPs and large indels without mapping, assembly or de Bruijn graphs.ResultsWhen applied to mosaic penicillin binding protein (PBP) genes in Streptococcus pneumoniae, we found near perfect concordance with assembled contigs at a fraction of the CPU time. Multilocus sequence typing (MLST) with this approach was able to bypass de novo assemblies. Kestrel has a very low false-positive rate when applied to the whole genome, and while Kestrel identified many variants missed by other methods, limitations of a purely k-mer based approach affect overall sensitivity.Availability and implementationSource code and documentation for a Java implementation of Kestrel can be found at https://github.com/paudano/kestrel. All test code for this publication is located at https://github.com/paudano/kescases.Supplementary information Supplementary data are available at Bioinformatics online.

Predominance of Serogroup 19 CC320/271 among Penicillin-Nonsusceptible Streptococcus pneumoniae Isolates after Introduction of the PCV7 Vaccine in Several Regions of Japan.

Multidrug-resistant Streptococcus pneumoniae serogroup 19, including serotypes 19A and 19F, associated with clonal complex 320/271 (CC320/271), has been previously shown to be predominant in many countries after introduction of a 7-valent pneumococcal conjugate vaccine (PCV7). However, in Japan there has been no epidemiological research focused on penicillin-nonsusceptible isolates after this event. Therefore, we aimed to characterize penicillin-nonsusceptible S. pneumoniae (PNSSP; penicillin minimum inhibitory concentration [MIC] ≥ 4.0 μg/ml) after the introduction of PCV7 in Japan. Throughout Japan, we collected 1,057 pneumococcal isolates from 2010 to 2014. We then evaluated MICs and performed serotyping, multilocus sequence typing, and sequencing of penicillin-binding protein genes in 51 isolates (penicillin MIC ≥ 2.0 μg/ml). Twenty-three isolates (2.2%) showed penicillin nonsusceptibility (penicillin MIC ≥ 4.0 μg/ml). Serotypes 19F (14 isolates, 60.9%) and 23F (4 isolates, 17.4%), which are covered by the vaccine, were predominant among PNSSP strains. Only 3 isolates belonged to nonvaccine serotype 19A. Among the PNSSP isolates, CC320/271 (16/23 strains, 69.6%) was the most prevalent clone. Moreover, CC320/271 clones showed high MIC values of a third-generation cephalosporin. Thus, we demonstrated clonal predominance of serogroup 19 CC320/271 with strong resistance to β-lactams including a third-generation cephalosporin among PNSSP isolates.

Impacts of Penicillin Binding Protein 2 Inactivation on β-Lactamase Expression and Muropeptide Profile in Stenotrophomonas maltophilia.

Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in ampR-β-lactamase module-harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA, mrcB, pbpC, mrdA, ftsI, dacB, and dacC, in the Stenotrophomonas maltophilia genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, S.maltophilia harbors two β-lactamase genes, L1 and L2, whose expression is induced via β-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of mrdA resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG, ampDI (the homologue of Escherichia coli ampD), nagZ, ampR, and creBC in L1/L2 expression mediated by a ΔmrdA mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain ΔmrdA-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant ΔmrdA-mediated L1/L2 expression relied on functional AmpNG, AmpR, and NagZ, was restricted by AmpDI, and was less related to the CreBC two-component system. Inactivation of mrdA significantly increased the levels of total and periplasmic N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamyl-meso-diamnopimelic acid-d-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain ΔmrdA-mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E.cloacae. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an mrdA deletion mutant of S.maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for ΔmrdA-mediated β-lactamase expression in S.maltophilia. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance.

New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary.

ABSTRACTThe Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murMHu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murMHu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2xHu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2xHu17 and pbp1a of strain Hu17 (pbp1aHu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient.

Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S.pneumoniae strains except for pbp1a of a Romanian S.pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S.pneumoniae to S.mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S.pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCEStreptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S.pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S.mitis as donor species. Sequences identical to one such mosaic gene were identified in an S.mitis strain from the same patient, suggesting that in this case S.pneumoniae played the role of donor.

Serotype distribution, antimicrobial resistance and prevalence of pilus islets in pneumococci following the use of conjugate vaccines.

In Japan, the 7-valent pneumococcal vaccine (PCV7) was introduced in 2010 and, in 2013, the PCV7 was replaced with the 13-valent pneumococcal vaccine (PCV13). This study was conducted to investigate serotypes, antimicrobial resistance and prevalence of pilus islets in pneumococcal isolates from inpatients in a Japanese tertiary hospital. From April 2011 to February 2016, 151 isolates [95 (18 children, 77 adults) and 56 (19 children, 37 adults) in the PCV7 and PCV13 periods, respectively] were collected. All isolates were serotyped using genetic methods and were tested for susceptibility to 18 antimicrobials. Unaltered penicillin-binding protein (PBP) genes, macrolide resistance genes and pilus islets were identified by PCR. Between the two periods, the prevalence of non-PCV13 serotypes was shown to increase from 50.0 to 78.9 % in children, and serotype 3 increased from 14.3 to 24.3 % in adults. Six of seven isolates from invasive diseases were assigned to non-PCV13 serotypes. Overall, multidrug resistance (MDR) was detected in 46.4 % of isolates, which included the dominant non-PCV13 serotypes 6E, 15A and 23A (prevalence≥75.0 %). gPRSP (three altered genes pbp1a, pbp2b and pbp2x) and macrolide resistance genes [erm(B) and/or mef(A/E)] were detected in 35.8 and 93.4 % of all isolates, respectively. Pilus islets [PI-1 (clade I, II and III) and/or PI-2] were found in 22.5 % (34/151) of isolates belonging to six different serotypes (19F, 23F, 19A, 6E, 15B and 35B) and 88.2 % (30/34) of these exhibited MDR. This study revealed the spread of MDR in several non-PCV13 serotypes and in isolates with pilus islets.

Impact of the pneumococcal conjugate vaccine on serotype distribution of adult non-invasive Streptococcus pneumoniae isolates in Tokai region, Japan, 2008-2016.

The introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in February 2010 markedly reduced the burden of invasive pneumococcal disease and changed serotype distribution in Japan. In November 2013, PCV7 was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13). We investigated the serotype distribution and susceptibility trends of non-invasive Streptococcus pneumoniae isolates collected from adult patients. A total of 504 pneumococcal isolates were collected during 4 periods between 2008 and 2016 (Period 1; between June 2008 and April 2009, Period 2; between September 2010 and March 2011, Period 3; between October 2011 and March 2012, Period 4; between August 2015 and January 2016). The coverage of PCV7 and PCV13 significantly decreased from 38.6% and 60.5% in Period 1 to 6.6% and 31.1% in Period 4. This change was mainly due to a large decrease in the frequency of serotype 19F, 6B, and 14. Serotype 3 was the most frequently isolated, and gradually increased. Additionally, non-PCV13 serotypes 11A, 33F, and 35B significantly increased. Most of the PCV7 serotypes 19F, 23F, 6B, and 14 had mutations of penicillin-binding protein genes and macrolide resistance genes, and these serotypes showed low susceptibilities to cefdinir and clarithromycin. On the other hand, a significant change in susceptibility to various antimicrobial agents was not observed throughout the study period, except for decreased susceptibility to carbapenems. Continuous surveillance studies of pneumococcal serotype changes and drug susceptibility are necessary in future.

Antimicrobial resistance, penicillin-binding protein sequences, and pilus islet carriage in relation to clonal evolution of Streptococcus pneumoniae serotype 19A in Russia, 2002–2013

Clonal changes of serotype 19A pneumococci have been appreciated in conjunction with growing prevalence of this serotype after implementation of the seven-valent pneumococcal conjugate vaccine (PCV7). In the present study, we characterized serotype 19A pneumococci collected in Russia within a decade preceding the implementation of PCV vaccination and described their clonal evolution. We retrospectively analyzed non-invasive serotype 19A isolates collected in 2002-2013. All isolates were subjected to multilocus sequence typing, antimicrobial susceptibility testing, determination of macrolide resistance genotype, molecular detection of pilus islet (PI) carriage, sequencing of penicillin-binding protein (PBP) genes. A total of 49 serotype 19A isolates represented 25 sequence types, of which 14 were newly described. The majority of isolates were distributed among clonal complex (CC) 663 (28%), CC230 (25%), CC156, and CC320 (14% each). CC663 and CC156 dominated in 2003, but were replaced by CC230 and CC320 later on; CC320 was only evident starting 2010. All isolates of CC663 and CC156 carried PI1; CC320 possessed both PI1 and PI2. The overall rate of altered amino acids in penicillin-nonsusceptible isolates was 13·9%, 7·2%, and 8·7% for PBP1a, PBP2b, and PBP2x, respectively. Our findings demonstrate that the clonal structure of serotype 19A pneumococci may evolve without PCV pressure.

Clinical Characteristics of Invasive Pneumococcal Disease of the Mucoid Phenotype：A Case Series

Streptococcus pneumoniae is a gram-positive coccus that causes invasive and non-invasive pneumococcal diseases. Invasive pneumococcal disease (IPD) is defined as an infection confirmed by the isolation of S. pneumoniae from a normally sterile site. IPD has a high mortality rate of around 20% in adults. Colonies of S. pneumoniae show various morphological features which include a mucoid appearance. The production of large amounts of capsular polysaccharide gives colonies of S. pneumoniae their mucoid appearance. This is thought to be a mechanism of microbial escape from phagocyte killing, leading to strong pathogenicity despite their high susceptibility to antibiotics. To our knowledge, the clinical characteristics of patients with mucoid IPD are unknown. We retrospectively analyzed the records of adult patients with IPD of the mucoid phenotype at Teine Keijinkai Hospital, Hokkaido, Japan, between 2009 and 2015. Seven patients (six males) with age range between 62 and 80 years were diagnosed as having mucoid IPD during the study period. Two patients had a history of malignancy and diabetes mellitus respectively, and six patients were living independently. No patients were previously vaccinated with pneumococcal vaccine. Six patients had clinical manifestations of pneumonia; one patient had multiple concomitant complications of mastoiditis, meningitis, and prosthetic joint infection. All isolates were isolated from blood. All isolates were susceptible to beta-lactam antibiotics, while six isolates were resistant to macrolides and chloramphenicol. Based on the Quellung reaction and real-time polymerase chain reaction (PCR) results, one isolate of mucoid phenotype was confirmed as serotype 3 with the penicillin binding protein gene of pbp2x and the macrolide resistant gene of ermB. Although all patients received appropriate antibiotics based on susceptibility testing, four patients required mechanical ventilation and vasopressors. One patient had neurological sequelae (hypacusis) and two patients died during the course of hospitalization. This case series suggests that mucoid IPD can occur in immunocompetent hosts and can cause high mortality. Since most of the mucoid phenotype of S. pneumoniae are serotype 3, widespread use of pneumococcal vaccine is important to prevent morbidity and mortality associated with IPD.

Exposure to β-lactams results in the alteration of penicillin-binding proteins in Clostridium perfringens

Clostridium perfringens causes a variety of mild to severe infections in humans and other animals. A decrease in the affinity of penicillin-binding protein (PBP) transpeptidases for β-lactams is considered one of the mechanisms of β-lactam resistance in bacteria. Two strains of C. perfringens isolated from bovines and one isolated from a chicken, which had decreased susceptibility to β-lactams, had variations in the amino acid sequences of the central penicillin-binding regions of the PBPs. β-Lactam-resistant mutants of another C. perfringens strain, ATCC 13124, were selected in vitro to determine the effects of exposure to β-lactams on the PBP genes. Cultures of the wild type rapidly developed resistance to penicillin G, cephalothin and ceftriaxone. The susceptibilities of all of the selected mutants to some other β-lactams also decreased. The largest PBP found in C. perfringens, CPF_2395, appeared to be the primary target of all three drugs. Strain resistant to penicillin G had mutation resulting in the substitution of one amino acid within the central penicillin-binding/transpeptidase domain, but the ceftrioxane and cephalothin-resistant strains had mutations resulting in the substitution of two amino acids in this region. The cephalothin-resistant mutant also had additional mutations in the CPF_0340 and CPF_2218 genes in this critical region. No other mutations were observed in the three other PBPs of the in vitro resistant mutants. Resistance development also altered the growth rate and cell morphology of the mutants, so in addition to the PBPs, some other genes, including regulatory genes, may have been affected during the interaction with β-lactam antibiotics. This is the first study showing the effects of β-lactam drugs on the substitution of amino acids in PBPs of C. perfringens and points to the need for studies to detect other unknown alterations affecting the physiology of resistant strains.

Four Cases of Infantile Otitis Media Caused by the Newer Quinolone-resistant and gBLNAR

We have encountered four cases of infantile otitis media caused by multidrug-resistant Haemophilus influenzae, including to the newer quinolones. The antimicrobial sensitivity of each bacterial isolate was tested on the basis of the minimum inhibitory concentration (MIC). Each isolate was then tested using the polymerase chain reaction (PCR) to investigate whether the TEM β-lactamase production gene was present, and to detect mutations in the penicillin-binding protein (PBP) genes and mutations leading to amino acid substitutions in the quinolone resistance-determining region (QRDR). The isolates were found to be classifiable into two types according to the amino acid-substitution mutations. Molecular epidemiological analysis by pulsed field gel electrophoresis (PFGE) revealed different electrophoretic patterns in all the H. influenzae isolates, suggesting that the four patients were infected by different strains.

Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6.

Penicillin-binding proteins (PBPs) are membrane-associated enzymes, which are involved in the last two steps of peptidoglycan biosynthesis, and some of them are key players in cell division. Furthermore, they are targets of β-lactams, the most widely used antibiotics. Nevertheless, very little is known about the expression and regulation of PBP genes. Using transcriptional mapping, we now determined the promoter regions of PBP genes from the laboratory strain Streptococcus pneumoniae R6 and examined the expression profile of these six promoters. The extended −10 region is highly conserved and complies with a σA-type promoter consensus sequence. In contrast, the −35 region is poorly conserved, indicating the possibility for differential PBP regulation. All PBP promoters were constitutively expressed and highly active during the exponential and early stationary growth phase. However, the individual expression of PBP promoters varied approximately fourfold, with pbp1a being the highest and pbp3 the lowest. Furthermore, the deletion of one nucleotide in the spacer region of the PBP3 promoter reduced pbp3 expression ∼10-fold. The addition of cefotaxime above the minimal inhibitory concentration (MIC) did not affect PBP expression in the penicillin-sensitive R6 strain. No evidence for regulation of S. pneumoniae PBP genes was obtained.

Impact of Nigella sativa and Clove Oils on Cell Wall Genes Expression in Multidrug Resistant Staphylococcus aureus

Antimicrobial resistance of methicillin resistant Staphylococcus aureus (MRSA) became on the top list of the serious problems that have a negative impact on public health. Therefore, this study aimed to determine the in vitro antibacterial activities of Nigella sativa (N. sativa) and clove essential oils against multidrug resistant (MDR) methicillin resistant Staphylococcus aureus (MRSA) isolates recovered from different sources with a special reference to their role on the expression of penicillin binding protein (pbp 2) and muscle ring finger (murF) genes. Antibiotic susceptibility testing of 51 staphylococcal isolates comprising 34 Staphylococcus aureus (S. aureus) and 17 coagulase negative staphylococci (CoNs) revealed a highest sensitivity against vancomycin (100%) and marked resistance patterns against β-lactam (beta-lactam) antibiotics. The results revealed that 97.1% of S. aureus isolates demonstrated a high level of MDR pattern, being resistant to more than 3 antibiotics of different classes. The in vitroantibacterial effects of clove and Nigella sativa essential oils against 11 MDR isolates using disc diffusion method indicated that both oils exhibited strong inhibitory efficiencies with inhibition zone diameters up to 45 and 20 mm, respectively. Besides, broth microdilution test of both essential oils revealed maximum activities against the tested strains with minimum inhibitory concentrations (MICs) up to 0.5 and 8 µg/mL for clove and N. sativa oils, respectively. Quantitative RT-PCR (real time polymerase chain reaction) analysis revealed the effective role of N. sativa and clove oils on the down-regulation of S. aureus murF and pbp 2 genes. In conclusion, the above findings highlight the promising antibacterial functions of N. sativa and clove essential oils in the treatment of emergent resistant S. aureus infections.

Molecular characteristics of penicillin-binding protein 2b, 2x and 1a sequences in Streptococcus pneumoniae isolates causing invasive diseases among children in Northeast China.

Streptococcus pneumoniae is one of the common pathogens causing severe invasive infections in children. This study aimed to investigate the serotype distribution and variations of penicillin-binding proteins (PBPs) 2b, 2x and 1a in S. pneumoniae isolates causing invasive diseases in Northeast China. A total of 256 strains were isolated from children with invasive pneumococcal disease (IPD) from January 2000 to October 2014. All strains were serotyped and determined for antibiotic resistance. The amplicons of penicillin-binding domains in pbp1a, pbp2b and pbp2x genes were sequenced for variation identification. The most prevalent serotypes of isolates in IPD children were 19A, 14, 19F, 23F and 6B. 19A and 19F were the most frequent serotypes of penicillin-resistant S. pneumoniae (PRSP), which present with high resistance to amoxicillin, cefotaxime, ceftriaxone and meropenem. The numbers of amino acid substitutions of penicillin-non-susceptible S. pneumoniae (PNSP) isolates were higher than those of penicillin-sensitive S. pneumoniae isolates in all the PBP genes (p < 0.01). The patterns of amino acid mutation in PBP2b, PBP2x and PBP1a were unique and different from those of other countries. All of the serotype 19A and 19F PRSP isolates carried 25 amino acid mutations, including Ala618 → Gly between positions 560 and 675 in PBP2b and Thr338 → Ala substitutions in PBP2x. The amino acid alterations in PBP2b, PBP2x and PBP1a from S. pneumoniae were closely associated with resistance to β-lactam antibiotics. This study provides new data for further monitoring of genetic changes related to the emergence and spread of resistance to β-lactam antibiotics in China.

Metabolic engineering of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid.

5-Aminolevulinic acid (5-ALA) has recently attracted attention for its potential applications in the fields of medicine and agriculture. In this study, Corynebacterium glutamicum was firstly engineered for 5-ALA production via the C4 pathway. HemA encoding 5-aminolevulinic acid synthase from Rhodobacter sphaeroides was codon optimized and expressed in C. glutamicum ATCC13032, resulting in accumulation of 5-ALA. Deletion of all known genes responsible for the formation of acetate and lactate further enhanced production of 5-ALA. Overexpression of ppc gene encoding phoenolpyruvate carboxylase resulted in an accumulation of 5-ALA up to 2.06 ± 0.05 g/L. Furthermore, deletion of high-molecular-weight penicillin-binding proteins (HMW-PBPs) genes pbp1a, pbp1b, and pbp2b led to an increase in 5-ALA production of 13.53%, 29.47%, and 22.22%, respectively. Finally, 5-ALA production was enhanced to 3.14 ± 0.02 g/L in shake flask by heterologously expressing rhtA encoding threonine/homoserine exporter, and 86.77% of supplemented glycine was channeled toward 5-ALA production in shake flask. The engineered C. glutamicum ALA7 strain produced 7.53 g/L 5-ALA in a 5 L bioreactor. This study demonstrated the potential utility of C. glutamicum as a platform for metabolic production of 5-ALA. Change of cell permeability by metabolic engineering HMW-PBPs may provide a new strategy for biochemicals production in Corynebacterium glutamicum. Biotechnol. Bioeng. 2016;113: 1284-1293. © 2015 Wiley Periodicals, Inc.

Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant.

Beta-lactam resistant clinical isolates of Streptococcus pneumoniae contain altered penicillin-binding protein (PBP) genes and occasionally an altered murM, presumably products of interspecies gene transfer. MurM and MurN are responsible for the synthesis of branched lipid II, substrate for the PBP catalyzed transpeptidation reaction. Here we used the high-level beta-lactam resistant S. oralis Uo5 as donor in transformation experiments with the sensitive laboratory strain S. pneumoniae R6 as recipient. Surprisingly, piperacillin-resistant transformants contained no alterations in PBP genes but carried murEUo5 encoding the UDP-N-acetylmuramyl tripeptide synthetase. Codons 83-183 of murEUo5 were sufficient to confer the resistance phenotype. Moreover, the promoter of murEUo5 , which drives a twofold higher expression compared to that of S. pneumoniae R6, could also confer increased resistance. Multiple independent transformations produced S. pneumoniae R6 derivatives containing murEUo5 , pbp2xUo5 , pbp1aUo5 and pbp2bUo5 , but not murMUo5 sequences; however, the resistance level of the donor strain could not be reached. S. oralis Uo5 harbors an unusual murM, and murN is absent. Accordingly, the peptidoglycan of S. oralis Uo5 contained interpeptide bridges with one L-Ala residue only. The data suggest that resistance in S. oralis Uo5 is based on a complex interplay of distinct PBPs and other enzymes involved in peptidoglycan biosynthesis.

Genetic analyses of penicillin binding protein determinants in multidrug-resistant Streptococcus pneumoniae serogroup 19 CC320/271 clone with high-level resistance to third-generation cephalosporins.

We describe the dissemination of a multidrug-resistant (MDR) serogroup 19 pneumococcal clone of representative multilocus sequence type 271 (ST271) with high-level resistance to cefotaxime in Hong Kong and penicillin binding protein (pbp) genes and its relationships to Taiwan(19F)-14 and the prevalent multidrug-resistant 19A clone (MDR19A-ST320). A total of 472 nonduplicate isolates from 2006 and 2011 were analyzed. Significant increases in the rates of nonsusceptibility to penicillin (PEN) (MIC ≥ 4.0 μg/ml; 9.9 versus 23.3%; P = 0.0005), cefotaxime (CTX) (MIC ≥ 2.0 μg/ml; 12.2 versus 30.3%; P < 0.0001 [meningitis MIC ≥ 1.0 μg/ml; 30.2 versus 48.7%; P = 0.0001]), and erythromycin (ERY) (69.2 versus 84.0%; P = 0.0003) were noted when rates from 2006 and 2011 were compared. The CTX-resistant isolates with MICs of 8 μg/ml in 2011 were of serotype 19F, belonging to ST271. Analyses of the penicillin binding protein 2x (PBP2x) amino acid sequences in relation to the corresponding sequences of the R6 strain revealed M339F, E378A, M400T, and Y595F substitutions found within the ST271 clone but not present in Taiwan(19F)-14 or MDR19A. In addition, PBP2bs of ST271 strains and that of the Taiwan(19F)-14 clone were characterized by a unique amino acid substitution, E369D, while ST320 possessed the unique amino acid substitution K366N, as does that of MDR19A in the United States. We hypothesize that ST271 originated from the Taiwan(19F)-14 lineage, which had disseminated in Hong Kong in the early 2000s, and conferred higher-level β-lactam and cefotaxime resistance through acquisitions of 19 additional amino acid substitutions in PBP2b (amino acid [aa] positions 538 to 641) and altered PBP2x via recombination events. The serogroup 19 MDR CC320/271 clone warrants close monitoring to evaluate its effect after the switch to expanded conjugate vaccines.

Genotypic characterization of Malaysian human isolates of Streptococcus pneumoniae from carriage and clinical sources.

This study characterized carriage and clinical pneumococcal isolates for serotypes, penicillin susceptibility, virulence genes and restriction fragment length polymorphism (RFLP) pattern of penicillin binding protein (PBP) genes. DNA fingerprint of isolates was generated by BOX-PCR. Majority of serotypes were 23F followed by 19F, 19A and 6A. Twenty-four percent of isolates were penicillin non-susceptible (PNSP). All of the targeted virulence genes were detected in all isolates with the exception of pili; 20.6% (n=22) for PI-1 and 14.0% (n=15) for PI-2. Of the 13 isolates which carried both PI-1 and PI-2, 10 were of clinical origin. Digested pbp-DNA produced three PBP-RFLP profiles for pbp1a (A1 to A3), six profiles for pbp2b (B1 to B6) and seven for pbp2x (X1 to X7) mostly in PNSPs. Based on BOX-PCR analysis, the majority of isolates were genetically diverse with a small number of potentially related isolates carrying pili genes. No obvious genotypic association was observed pertaining to carriage and clinical origin of isolates.

Growth of Streptococcus pneumoniae on Macconkey Agar: Possibility of Impossible?

Dear Editor Streptococcus pneumoniae (S. pneumoniae) is Gram-positive lanceolate-shaped diplococci which is a significant human pathogenic bacterium causing pneumonia, meningitis and various other infections. It is a fastidious organism and grows well in 5% carbondioxide (CO2) and media containing blood (blood agar, chocolate gar) as growth requires a source of catalase (e.g. blood) to neutralize the large amount of hydrogen peroxide (H2O2) produced by the bacteria [1]. Hence, it does not grow on MacConkey agar (MA) due to the absence of blood as well as due to the presence of high concentration of bile in the medium which is inhibitory to the growth of S. pneumoniae. However, to the best of our knowledge, we are reporting the first and unusual case of S. pneumoniae growth on MA. We received cerebrospinal fluid from a 21-year-old male patient who was admitted with fever, headache, vomiting and clinically had signs of meningitis. Gram stain revealed few pus cells. Biochemical parameters showed decreased glucose, moderately increased protein. Culture revealed pure growth of tiny opaque lactose-fermenting colonies on MA and alpha-haemolytic colonies on blood agar (BA) and chocolate agar (CA). Colony smear from all the three plates showed lanceolate-shaped Gram positive cocci in pairs and short chains. Identification and antibiotic sensitivity testing was done by Vitek-2 compact (BioMerieux) using GPC identification card (GP 21 342) and Streptococcal AST card (AST-ST01) respectively. The organism was identified as Streptococcus pneumoniae which was sensitive to all the antibiotics tested except benzylpenicillin. However, the growth of the isolate on MA created a dilemma and hence the same was sent for identification by MALDI-TOF method. The later identified the isolate as Streptococcus pneumoniae with score value of 2.114. Several studies have highlighted evolving clinical problems with S. pneumoniae. Increasing resistance to antimicrobial agents, and failure of traditional optochin(ethylhydrocupreine) sensitivity had been a growing concern [2]. Dias CA et al., reported the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase [3]. Clinical isolate with intermediate sensitivity to penicillin was first reported in 1967. Since then, penicillin non-susceptible S. pneumoniae has been reported with increasing frequency in many countries.The resistance to penicillin arises from mosaic mutation of penicillin binding protein (PBP) genes due to interspecies recombination of homologous genes [4]. Growth of S. pneumoniae on MA may be a beginning of the changing trend of the organism’s fastidious growth requirements. One of the possible explanations for the same may be some mutation causing alteration in hydrogen peroxide producing capacity of S. pneumoniae and hence bypassing the need for catalase in the media used.Some strains of S. pneumoniae with such mutations have been used in experimental studies to create an environment with low concentration of H2O2 [5,6]. Pericone et al., used loss-of-function mutations of spxB gene in P62 and P878 strains of S. pneumoniae to study the effect of low H2O2 on rate of spontaneous gene mutation. The lower spxB expression resulting in under-expression of pyruvate oxidase results in the lower production of hydrogen peroxide. A spxB-defective pneumococcal mutant produces opaque colony and opaque variants show increased virulence for non-nasopharyngeal infections [5]. In our case also the colony morphology was opaque on MA, and the infection site was meninges. So, these findings provides an assumption that the spx-B mutation can be occurring in the clinical isolates like in our case thus leading to diminished production of hydrogen peroxide and hence not requiring catalase in the growth medium. Due to unavailability of molecular methods at our institution the genetic analysis could not be performed in this case. However, it is advisable that in future if any such scenario is encountered by any institution the genetic analysis could be of great help.

Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene.

ObjectivesMethicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to β-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to β-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant β-lactam antibiotics.MethodsThe four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays.ResultsWe identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types.ConclusionsThis work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for β-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.