Pbp Genes Research Articles

Epidemiological and molecular characterization of invasive Streptococcus pneumoniae isolated following introduction of 7-valent conjugate vaccine in Kinki region, Japan, 2008–2013

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Heterogeneity of penicillin-non-susceptible group B streptococci isolated from a single patient in Germany.

Objectives Streptococcus agalactiae [group B streptococci (GBS)] have been considered uniformly susceptible to penicillin. However, increasing reports from Asia and North America are documenting penicillin-non-susceptible GBS (PRGBS) with mutations in pbp genes. Here we report, to the best of our knowledge, the first two PRGBS isolates recovered in Europe (AC-13238-1 and AC-13238-2), isolated from the same patient. MethodsTwo different colony morphologies of GBS were noted from a surgical abscess drainage sample. Both were serotyped and antimicrobial susceptibility testing was performed by different methodologies. High-throughput sequencing was done to compare the isolates at the genomic level, to identify their capsular type and ST, to evaluate mutations in the pbp genes and to compare the isolates with the genomes of other PRGBS isolates sharing the same serotype and ST.ResultsIsolates AC-13238-1 and AC-13238-2 presented MICs above the EUCAST and CLSI breakpoints for penicillin susceptibility. Both shared the capsular type Ia operon and ST23. Genomic analysis uncovered differences between the two isolates in seven genes, including altered pbp genes. Deduced amino acid sequences revealed critical substitutions in PBP2X in both isolates. Comparison with serotype Ia clonal complex 23 PRGBS from the USA reinforced the similarity between AC-13238-1 and AC-13238-2, and their divergence from the US strains.ConclusionsOur results support the in-host evolution of β-lactam-resistant GBS, with two PRGBS variants being isolated from one patient.

615. Daptomycin (DAP) Synergy with β-Lactams in DAP-Resistant (DAP-R) E. faecium (Efm) Is Dependent On PBP5 Sequence and β-Lactam-binding Affinity

BackgroundDAP in combination with β-lactams is a viable option to treat recalcitrant DAP-R/tolerant strains of Efm. Ampicillin (AMP), ceftaroline (CPT), and ertapenem (ERT) have the best synergism. Using a DAP tolerant strain (503; DAP MIC 2 µg/mL) of Efm, we previously showed that AMP, CPT, and ERT combined with DAP were effective in reducing bacterial loads and prevented emergence of resistance in a simulated endocardial vegetation model. However, against a DAP-R Efm strain (R497, DAP MIC of 16 µg/mL), CPT, ERT failed to synergize with DAP. Here, we dissect the mechanistic basis of the differing DAP plus β-lactam synergistic effect.MethodsWe performed comparative transcriptional profiling of pbp genes in Efm 503 vs. R497 using qRT–PCR. PBP5 protein levels were assessed by immunoblotting. The β-lactam-binding affinity of PBPs was quantified with bocillin-FL staining and SDS–PAGE. PBP5 sequences of Efm Com15 (AMP and DAP-susceptible strain) and clinical strains S447, 503 and R497 (all with AMP MIC > 256 µg/mL) were compared in silico to identify amino acid (AA) differences in key protein sites which were verified with sequencingResults Pbp gene transcripts and PBP5 amounts were similar between 503 vs. R497. Interestingly, bocillin SDS–PAGE showed increased β-lactam binding affinity in PBP5 of 503 compared with that of R497 and S447. PBP5 sequences of S447 and R497 were identical. All three clinical strains had classic mutations (M485A and 466’S) important for high-level AMP-R. However, 503 had additional substitutions in the transpeptidase domain (H408Q, A462V, T546N, T558A, S582G, V586L) and penicillin-binding domain (Q632K, L642P) compared with R497 and S447. The latter AA sequences in 503 are common to AMP-susceptible Efm strainsConclusionWe uncovered that a “hybrid” pbp5 allele of 503 (DAP-tolerant) correlated with synergism of DAP plus AMP, CPT or ERT and was associated with increased PBP5 β-lactam binding affinity. Lack of synergism of DAP plus CPT or ERT is associated with specific PBP amino acids in the transpeptidase and penicillin-binding domains. Thus, pbp5 alleles are major determinants of the DAP plus β-lactam synergistic effect and could be used as a diagnostic tool to guide therapy in recalcitrant Efm infectionsDisclosures All authors: No reported disclosures.

Whole-genome sequencing of Staphylococcus aureus L401, a mecA-negative community-associated methicillin-resistant strain isolated from a healthy carrier.

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen of great concern owing to its antimicrobial resistance and virulence properties. Here we report the first draft genome sequence of a mecA-negative community-associated MRSA strain isolated from a healthy young Mexican paediatric carrier in order to reveal the genomic structure underlying the multidrug-resistant phenotype and to discover the virulence properties of this strain. The draft genome sequence of S. aureus L401 was obtained using an Ion Torrent™ PGM platform. De novo assembled contigs were annotated, and antimicrobial resistance genes and virulence factors were identified using ResFinder and VirulenceFinder, respectively. In addition, a mutational survey of native pbp, gdpP and yjbH genes was performed. In silico multilocus sequence typing (MLST) and spa typing were also performed. S. aureus L401 has a genome size of 2 831 587 bp with 2799 protein-coding sequences. Various antimicrobial resistance genes conferring resistance to aminoglycosides, β-lactams, fluoroquinolones and macrolide-lincosamide-streptogramin B antimicrobials were found. Although both mecA and staphylococcal cassette chromosome mec (SCCmec) elements were absent, a missense mutation in PBP3 was identified. Moreover, genes encoding exfoliative toxin A, γ- and β-haemolysin, and several enterotoxins were also identified. S. aureus L401 belongs to ST109 and spa type t209. The availability of this genome will allow an insight into S. aureus resistance and virulence determinants as well as its epidemiology, lineage, evolution and genomic features involved in the paediatric commensal carriage.

Penicillin Binding Proteins3 and 4 Relations between Resistance Phenotypes and mecA, TEM genes expression in Staphylococcal aureus

This study was to determine the relationship between pbp3 and pbp4 gene compared with mecA and TEM resistance genes expression patterns. Total 134 clinical S. aureus strains were subjected to 19 antimicrobial susceptibility tests. We detected resistance to methicillin (mecA), penicillin (blaTEM) and expression of pbp (Penicillin-binding proteins) genes. We were compared blaTEM, extended spectrum, carbapenem related genes and types of SCCmec identified. Total of 134 clinical S. aureus strains, 79 (58.96%) in methicillin resistance, and 77 strains carried mecA. Prevalence rates of blaTEM and pbp genes were 107/134 (79.85%) and 128/134 (95.52%). Multiplex PCR results revealed that the predominant SCCmec type among 77 mecA-positive MRSA strains were similer too SCCmec type II 41.56% (32/77) and type IVA 40.26% (31/77). Prevalence rates of type IVb, IVd and non-typable were 18.18% (14/77), respectively. From a total of 77/134 (57.46%) MRSA isolate strains, 35/77 (45.46%) were positive for extended spectrum, 40/77 (51.95%) for cephalosporins, and 35/77 (45.46%) for carbapenems. The predominant SCCmec type II had more carbapenem resistances than IVA, IVb and IVd. TEM and mecA gene expression were not correlated with pbp gene, and the properties of drug resistance were appeared not associated with pbp3, 4 genes.

Isolation of group B Streptococcus with reduced β-lactam susceptibility from pregnant women

ABSTRACT β-Lactam antibiotics are first-line agents for the treatment and prevention of group B Streptococcus (GBS) infections. We previously reported clinical GBS isolates with reduced β-lactam susceptibility (GBS-RBS) and characterized them as harbouring amino acid substitutions in penicillin-binding proteins (PBPs). However, to our knowledge, GBS-RBS clinical isolates have never previously been isolated from pregnant women worldwide. We obtained 477 clinical GBS isolates from vaginal/rectal swabs of 4530 pregnant women in Japan. We determined the MICs of seven β-lactams for all 477 clinical isolates. Five clinical isolates showed reduced ceftibuten susceptibility. For these isolates, we performed sequencing analysis of pbp genes. None of the 477 isolates were non-susceptible to penicillin G, ampicillin, and meropenem. For five isolates, the MICs of ceftibuten were relatively high (64–128 μg/ml). Each of these isolates possessed a single amino acid substitution in PBP2X, and some of the substitutions had been previously found in GBS with reduced penicillin susceptibility. This is the first report of the isolation of clinical GBS-RBS isolates harbouring amino acid substitutions in PBP2X that confer reduced ceftibuten susceptibility from pregnant women.

Resistance to β-lactams in enterococci

Enterococci are intrinsically resistant to several antimicrobial classes and show a great ability to acquire new mechanisms of resistance. Resistance to β-lactam antibiotics is a major concern because these drugs either alone or in combination are commonly used for the treatment of enterococcal infections. Ampicillin resistance, which is rare in Enterococcus faecalis, occurs in most of the hospital-associated Enterococcus faecium isolates. High-level resistance to ampicillin in E. faecium is mainly due to the enhanced production of PBP5 and/or by polymorphisms in the beta subunit of this protein. The dissemination of high-level ampicillin resistance can be the result of both clonal spread of strains with mutated pbp5 genes and horizontal gene transfer.

Spermine and oxacillin stress response on the cell wall synthesis and the global gene expression analysis in Methicillin-resistance Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly emerging bacteria causing infection, which has developed resistance to most of the beta-lactam antibiotics because of newly acquired low-affinity penicillin-binding protein (PBP2a), which can continue to build the cell wall when beta-lactams block other PBPs. Exogenous spermine exerts a dose-dependent inhibition effect on the growth of Escherichia coli, Salmonella enterica serovar, and S. aureus. Selection of an MRSA Mu50 derivative which harbors mutation on PBP2 gene (named as MuM) showing spermine resistance and which confers a complete abolishment of spermine-beta-lactam synergy was identified. To further investigate the gene expression changes, a transcriptome profiling of MuM against Mu50 (wild-type) without any treatment, MuM and Mu50 in response to high dose spermine and Mu50 in response to spermine-beta-lactam synergy at 15, 30 and 60min time points was performed. Functional annotation was further performed to delineate the metabolic pathways associated with the significant genes. A significant down-regulation in the iron regulatory system, potassium channel uptake and polyamine transport system with an up-regulation in general stress response sigB dependent operon in MuM strain at 15, 30 and 60min time points with spermine treatment compared to Mu50 strain was observed. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion pattern of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. A strong relation between PBP2 protein and general stress sigB response, iron, potassium and polyamine transport systems was observed. SigB regulon should be activated on stress, which was not seen in some of our previous studies where it was down-regulated in wild-type Mu50 strain with spermine stress. Here, an intriguing finding is made where there seems to be a correction of this abnormal response of no SigB induction to a significant induction by PBP2 mutation. In MuM strain, a significant down-regulation of KdpABC operon genes at 15, 30 and 60min time points on spermine stress is seen, which seems to be absent without spermine treatment. Since KCL has been found to protect the cell against spermine stress in wild-type strain by induction of KdpABC operon, it fails to do so in MuM strain underlying the importance of PBP2 protein in spermine stress. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion patterns of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. Furthermore, spermine does not help in enhancing the binding of beta-lactams to PBPs and binding of spermine to PBPs does not cause conformational changes to PBPs, as tested with trypsin digestion patterns. Future studies on the molecular mechanism of spermine interactions with these systems hold great potential for the development of new therapeutics for MRSA infections.

Characteristics of Streptococcus pneumoniae serotype 19A isolates from children in the pre and post Conjugate Vaccine Era. Single center experience 1986–2015

BackgroundThe present study assessed the prevalence and characteristics of S. pneumoniae serotype 19A isolates from children with pneumococcal disease (PD), before and since introduction of pneumococcal conjugate vaccines (PCVs) in Greece. MethodsS. pneumoniae isolates collected at one large pediatric hospital between 1986 and 2015 were serotyped by the Quellung reaction and MICs determined by Etest. Alterations of pbp genes and the presence of mefA, mefE, ermB genes were detected by polymerase chain reaction. Genotypes were assessed by multilocus sequence typing (MLST). ResultsAmong 1875 isolates, 210 (11.2%) belonged to serotype 19A. The prevalence of PD caused by serotype 19A increased from 4.6% in the pre-PCV7 years (1986–2005) to 19.6% in the post-PCV7 years (2006–2010), peaking at 27% in 2009 (p < 0.001, 95% CI; 2.0, 18.2) with a significant upward trend (p = 0.04, 95% CI; 1.02, 12.66). Following the introduction of PCV13 in 2010, the rate decreased from 22% in 2011 to 11.4% in 2015 (p = 0.08, 95% CI; 0.92, 5.1) with a downward trend of borderline significance (p = 0.05, 95% CI; −6.8, 0.04). The multidrug resistant (MDR) serotype 19A isolates increased from 10.6% in 1986–2005 to 21.2% in 2006–2010 and to 71.8% in 2011–2015 (P < 0.001). Alterations in pbp genes were detected in all penicillin non-susceptible isolates. Of 110 erythromycin resistant isolates, 21 contained the mefE gene, 36 the ermB and 53 both the mefE and ermB genes. MLST analysis of 142 isolates revealed four dominant clonal complexes (CC); CC320, CC172, CC276 and CC199. The majority of CC320 and CC276 isolates displayed MDR phenotypes. ConclusionPD caused by serotype 19A increased significantly after the introduction of PCV7 followed by a decline after PCV13 use. The vast majority of persisting 19A isolates was MDR. Surveillance studies are necessary to monitor the changes in the pneumococcal population.

In vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during a national survey conducted in Belgian hospitals

OBJECTIVES The aim of this study was to estimate the in vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during national surveillance in Belgian acute-care hospitals. Ceftaroline-resistant isolates were further investigated for their resistance mechanisms. METHODS From October 2013 to March 2014, 155 laboratories of Belgian acute-care hospitals were invited to send to the National Reference Centre-Staphylococcus aureus (Belgium) up to five non-duplicate S. aureus including three MRSA and two MSSA from hospitalized patients. Isolates were analysed by spa typing, SCCmec typing (for MRSA) and PCR for detection of 16S-mecA-nuc and 16S-mecC. MICs of oxacillin, cefoxitin and ceftaroline were determined by the broth microdilution method. The nucleotide sequences of mecA, native pbp and gdpP genes of isolates with reduced susceptibility to ceftaroline were analysed for the presence of mutations responsible for amino acid substitutions. RESULTS Ninety-nine percent of isolates, including MRSA (n = 284) and MSSA (n = 131), were susceptible to ceftaroline. Only four MRSA isolates showed resistance to ceftaroline (MIC = 2 mg/L). These four isolates belonged to lineages CC5 (n = 1), CC22 (n = 2) and CC8 (n = 1). Two isolates (CC22 and CC8) carried mutations in mecA, as well as in other pbp genes. The remaining isolates carried mutations in native pbp genes or in gdpP. CONCLUSIONS This is the first Belgian in vitro survey on ceftaroline activity against S. aureus. This antibiotic showed excellent activity against MRSA and MSSA, and only a few MRSA isolates with resistance were found. Reduced susceptibility to ceftaroline seems a complex phenomenon due to the accumulation of mutations in genes involved in β-lactam tolerance.

Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in Enterococcus faecalis.

ABSTRACTEnterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 −35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.

Borderline oxacillin-resistant Staphylococcus aureus (BORSA) - a more common problem than expected?

Borderline oxacillin-resistant Staphylococcus aureus (BORSA) represents a quite poorly understood and inadequately defined phenotype of methicillin resistance. BORSA strains show low, borderline resistance to penicillinase-resistant penicillins (PRPs), with oxacillin MICs typically equal to 1-8 µg ml-1, and in contrast to methicillin-resistant S. aureus (MRSA), do not have an altered penicillin-binding protein, PBP2a, encoded by the mecA or mecC gene. Their resistance is typically associated with hyperproduction of beta-lactamases or, in some cases, point mutations in PBP genes. BORSA cannot be classified as either truly methicillin-resistant or truly methicillin-susceptible strains. However, they are frequently misidentified, which poses an obvious epidemiological and therapeutic threat. BORSA strains are commonly isolated from humans and animals, and are found both in hospitals and in a community setting. The epidemiology and clinical presentation of BORSA infections seem to be similar to those for MRSA; these infections are usually more severe than those caused by methicillin-sensitive S. aureus (MSSA). Treatment of severe infections caused by BORSA may be ineffective, even with larger doses of oxacillin. The available evidence suggests that BORSA represent a frequently neglected problem, and their emergence in new environments implies that they need to be monitored and accurately distinguished from MSSA and MRSA.

Molecular characterization, expression pattern and ligand‐binding properties of the pheromone‐binding protein gene from Cyrtotrachelus buqueti

AbstractPheromone‐binding proteins (PBPs) play important roles in the information exchange between insect sexes, specifically in the process of transporting fat‐soluble odour molecules from the external environment to olfactory receptors through the olfactory sensillum lymph. The PBP functions in this process may explain the sex pheromone identification mechanism used by insects, laying a theoretical foundation for the prevention and control of pests by interfering with olfactory recognition. In the present study, a PBP gene of Cyrtotrachelus buqueti (GenBank accession number: KU845733) is cloned for prokaryotic expression. Using N‐phenyl‐1‐naphthylamine as the fluorescent probe in a competitive binding assay, the ability of CbuqPBP1 to bind 12 sex pheromone analogues and three volatiles of Neosinocalamus affinis shoots is examined. Of the 12 C. buqueti sex pheromone analogues, dibutyl phthalate gives the greatest displacement (inhibitory constant value of 11.1 μm), whereas the other sex pheromone components show much smaller displacements. Consistent with other PBPs, the three plant volatiles (linalool, benzaldehyde and indole) show only a limited displacement of CbuqPBP1. However, the binding abilities of 1 : 1 ratios of each of the three plant volatiles with dibutyl phthalate show increases of 62.3%, 65.1% and 51.7% over the binding abilities of the three plant volatiles alone. CbuqPBP1 has dual roles in the processes of sensing sex pheromones and plant volatiles.

Characterization of high level ampicillin- and aminoglycoside-resistant enterococci isolated from non-hospital sources.

High level ampicillin- and aminoglycoside-resistant enterococci are being increasingly reported from non-hospital sources. This study was carried out to characterize these strains from non-hospital sources in Nigeria. A collection of Enterococcus faecium isolated from vegetables, soil, farm animals and manure and observed to be resistant to ampicillin (n=63) and gentamicin (n=37) discs, were screened for resistance to high levels of ampicillin and aminoglycoside using E-test strips. Putative high level ampicillin- and aminoglycoside-resistant strains were screened for pbp5 and aminoglycoside modifying enzyme genes, respectively, by PCR. The C-terminal region of the amplified pbp5 gene was also sequenced. Five (5/63) and thirty-five (35/37) of the ampicillin- and aminoglycoside-resistant strains were identified as high level ampicillin- and aminoglycoside-resistant E. faecium strains, respectively, based on the MIC results. The amplified pbp5 gene from the high level ampicillin-resistant isolates displayed 96-99 % nucleotide sequence similarity with the reference strains and three novel insertions (500Glu→Leu, 502Asp→Arg and 614Ile→Phe) in the amino acid sequence. Aminoglycoside modifying enzyme genes aac(6')-Ie-aph(2″) (100 %), aph(2')-Ic (88.8 %), aph(3')-IIIa (90 %) and ant(4')-Ia (40 %) were detected among the high level aminoglycoside-resistant isolates. This is the first report on the characterization of high level ampicillin- and aminoglycoside-resistant Enterococcus faecium among animals and vegetables in Nigeria. The results show that non-hospital sources can constitute a reservoir for potential dissemination of these strains and genes to humans via the food chain or by direct contact.

Genetic Characterization of vanA-Enterococcus faecium Isolates from Wild Red-Legged Partridges in Portugal.

Vancomycin-resistant enterococci (VRE) have been detected in wild animals representing a public health concern. The red-legged partridge (Alectoris rufa) is a common game bird and its meat is consumed in several countries, including Portugal. Three hundred five fecal samples of red-legged partridge from the north of Portugal were screened for VRE. Samples were cultured on Slanetz-Bartley agar supplemented with vancomycin (4 mg/L) and six vanA-Enterococcus faecium were recovered. Isolates were tested for antibiotic resistance and virulence genes. Multilocus sequence typing (MLST) was performed to study the genotypic diversity of vanA-containing E. faecium. The six isolates showed erythromycin resistance and harbored the erm(B) gene and the four that were tetracycline resistant showed the tet(M) gene. The C-terminal region of the pbp5 gene of the ampicillin-resistant isolates (minimal inhibitory concentration range of 256 μg/ml) was sequenced. Two different pbp5 alleles were detected when considering the changes of amino acid in 461-629 region. All isolates harbored the esp gene, whereas hyl, together with the esp gene, was detected in five isolates. MLST analysis grouped the isolates as ST448 (n = 1), ST139 (n = 1), and ST18 (n = 4). Our findings show that the red-legged partridges could be a reservoir of antimicrobial resistance genes and may contribute to the dissemination and transference of the resistance genes to other animals and humans.

Analysis of Point Mutations in the pbp2x, pbp2b, and pbp1a Genes of Streptococcus agalactiae and Their Relation with a Reduced Susceptibility to Cephalosporins.

The emergence of group B Streptococcus (GBS) isolates with reduced penicillin susceptibility (PRGBS) and their tendency to be nonsusceptible to fluoroquinolones prompted us to analyze the possible presence of amino acid mutations in penicillin-binding proteins (PBPs) (PBP2X, PBP1A, and PBP2B) from a collection of fluoroquinolone-resistant GBS isolates. We analyzed 21 GBS isolates resistant to levofloxacin. Sequence analysis of genes for PBPs was performed. The minimal inhibitory concentrations (MICs) for penicillin, ceftibuten, cefaclor, cefixime, cefotaxime, and ceftizoxime were performed by the Etest method and by broth microdilution method. The isolates were furthermore characterized by PCR-based capsular typing and analysis of surface protein genes. Genetic relatedness among the isolates was examined by multilocus sequence typing. Phylogenetic analysis of PBPs sequences was performed by Molecular Evolutionary Genetics Analysis software (MEGA7). All isolates were susceptible to penicillin, even if different mutations were detected in all PBPs in most of the isolates (12/21, 57%). However, we observed a reduced susceptibility to cefixime in seven isolates and to cefaclor in six isolates. These PSGBS isolates shared an I377V mutation in PBP2X and a T145A mutation in PBP1A. Most of the isolates belongs to the clonal complex 1, has serotype III and rib as surface protein. The results of phylogenetic comparative analysis show that several genetic lineages of our isolates with reduced susceptibility to cefixime/cefaclor have been independently emerging through the accumulation of mutations in their pbp genes, especially in pbp1a. If the MICs of penicillins and cephalosporins for GBS increase, careful epidemiological surveillance on this issue is recommended.

Characteristics of Streptococcus pneumoniae Serotype 19A Isolates from Children in the Pre- and Post-Conjugate Vaccine Era

Background The aim of the present study was to assess the prevalence and characteristics of S. pneumoniae serotype 19A isolates from invasive and non-invasive pneumococcal disease (IPD) in children, before and after the introduction of pneumococcal conjugate vaccines (PCVs) in Greece.Methods All S. pneumoniae isolates collected between 1986 and 2015 were re-cultured for further testing. Serotyping was performed using the quellung reaction and antibiotic susceptibilities were determined by E-test. The altered pbp genes (pbp2b, pbp2x, and pbp1a) conferring resistance to penicillin and the macrolide-resistant genes [mef(A), mef(E), and erm(B)] were detected by polymerase chain reaction (PCR) using specific primers. The sequence types (ST) and clonal complexes were determined by multilocus sequence typing (MLST).ResultsAmong 1,875 isolates examined, 210(11.2%) (belonged to serotype 19A. The proportion of serotype 19A isolates increased from 4.6% (47/1018) in the pre-PCV7 years (1986–2005) to 19.6% (88/449) in the post-PCV7 years (2006–2010) and to 18.4% (75/408) in the post PCV-10 and PCV13 years (2011–2015).The resistance frequencies to penicillin, cefotaxime, erythromycin, clindamycin, and tetracycline increased significantly after 2005 as compared with the preceding period (1986–2005) from 8.5% to 31.9%, 2.1% to 20.8%, 10.6% to 65.6%, 4.2% to 47.7% and 19.1% to 46.0%, respectively (P < 0.01). Moreover, 94 (57.7%) of 163 isolates after 2005 exhibited multidrug-resistant (MDR) phenotype, whereas only seven (15.9%) of 47 isolates from period 1986–2005 were MDR (P <0.001). All the penicillin non-susceptible isolates examined (N = 136), had altered one or more of the pbp genes. Of 112 erythromycin-resistant isolates, 22 contained the mefE gene, 39 the ermB and 51 both the mefE and ermB genes. MLST analysis of 31 invasive isolates revealed four clonal complexes (CC); CC172 (n = 11), CC199 (n = 8), CC320 (n = 5), and CC276 (n = 3), and four isolates belonged to CC2669, CC177, CC81, and CC63. The CC172 and CC199 were present throughout the study period, whereas the CC320 and CC276 appeared after 2009.Conclusion S. pneumoniae serotype 19A increased significantly after the introduction of PCV7 and a substantial proportion of these isolates exhibited MDR phenotype. The majority of the examined isolates belonged to limited number of CC.Disclosures V. Syriopoulou, Hellenic Center for Disease Control and Prevention: Grant Investigator, Research grant. Pfizer Inc.Pharmaceutical Company: Grant Investigator, Research grant

Different Vancomycin-Intermediate Staphylococcus aureus Phenotypes Selected from the Same ST100-hVISA Parental Strain.

The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.

Co-diversification of Enterococcus faecium Core Genomes and PBP5: Evidences of pbp5 Horizontal Transfer

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18, and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180–280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

Sex pheromone recognition and characterization of three pheromone-binding proteins in the legume pod borer, Maruca vitrata Fabricius (Lepidoptera: Crambidae).

Pheromone-binding proteins (PBPs) are essential for the filtering, binding and transporting of sex pheromones across sensillum lymph to membrane-associated pheromone receptors of moths. In this study, three novel PBP genes were expressed in Escherichia coli to examine their involvement in the sex pheromone perception of Maruca vitrata. Fluorescence binding experiments indicated that MvitPBP1-3 had strong binding affinities with four sex pheromones. Moreover, molecular docking results demonstrated that six amino acid residues of three MvitPBPs were involved in the binding of the sex pheromones. These results suggested that MvitPBP1-3 might play critical roles in the perception of female sex pheromones. Additionally, the binding capacity of MvitPBP3 with the host-plant floral volatiles was high and was similar to that of MvitGOBP2. Furthermore, sequence alignment and docking analysis showed that both MvitGOBP2 and MvitPBP3 possessed an identical key binding site (arginine, R130/R140) and a similar protein pocket structure around the binding cavity. Therefore, we hypothesized that MvitPBP3 and MvitGOBP2 might have synergistic roles in binding different volatile ligands. In combination, the use of synthetic sex pheromones and floral volatiles from host-plant may be used in the exploration for more efficient monitoring and integrated management strategies for the legume pod borer in the field.