An alternative penicillin-binding protein involved in Salmonella relapses following ceftriaxone therapy.

BACKGROUND: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy. METHODS: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2SAL or PBP3SAL. Affinity of PBP2SAL and PBP3SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model. FINDINGS: S. Typhimurium does not express PBP2SAL or PBP3SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3SAL was unable to cause relapse in mice following ceftriaxone therapy. INTERPRETATION: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3SAL, remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3SAL with novel drugs.

Characterisation of mecA gene negative Staphylococcus aureus isolated from bovine mastitis milk from Northern Germany.

Staphylococcus aureus (S. aureus) is an important causative agent of contagious intermammary infections in dairy cattle. S. aureus is also considered as an important foodborne pathogen and cause of food poisoning cases and outbreaks worldwide. In order to understand the molecular ecology of S. aureus, the present study compared phenotypic and genotypic characteristics of 70 S. aureus isolates from bovine mastitis milk samples collected during the period from August 2001 to March 2014 in different regions of Northern Germany. The S. aureus isolates were characterised phenotypically, as well as genotypically for their genetic diversity using multi-locus sequence typing (MLST), spa typing and the presence of virulence genes encoding 16 staphylococcal enterotoxins (sea-selu), toxic shock syndrome toxin (tst), thermonuclease (nuc), clumping factor (clfA and clfB), coagulase (coa) and the methicillin resistance gene mecA. A total of 16 sequence types were grouped into eight clonal complexes (CCs), and 17 spa types were identified. These included six novel sequence types and one novel spa type. The majority of bovine mastitis milk-associated sequence types belonged to the clonal complex CC5, CC97, CC133, and CC151 and showed closely related genotypes or lineages with sequence types of human origin. The genotype CC133 (ST133-t1403) was predominant, constituting 27.1% of the isolates. In addition, the S. aureus isolates displayed nine different enterotoxigenic profiles. All S. aureus were methicillin-susceptible (MSSA). The current study provides new information on phenotypic and genotypic traits of S. aureus isolates from bovine mastitis. The comparison of characteristics of isolates from the present study originating from mastitis milk showed similarities with human isolates. This might help to better understand the distribution of S. aureus in the one health context.

Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of ß-Lactamase-Producing Escherichia coli.

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing ß-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

Loss of a Class A Penicillin-Binding Protein Alters ß-Lactam Susceptibilities in Mycobacterium tuberculosis.

Recent studies have renewed interest in ß-lactam antibiotics as a potential treatment for Mycobacterium tuberculosis infection. To explore the opportunities and limitations of this approach, we sought to better understand potential resistance mechanisms to ß-lactam antibiotics in M. tuberculosis. We identified mutations in the penicillin-binding protein (PBP) ponA2 that were able to confer some degree of resistance to the cephalosporin subclass of ß-lactams. Surprisingly, deletion of ponA2 also confers resistance, demonstrating that ß-lactam resistance can spontaneously arise from PBP loss of function. We show that ponA2 mutants resistant to the cephalosporin subclass of ß-lactams in fact show increased susceptibility to meropenem, a carbapenem that is known to target l,d-transpeptidases, thereby suggesting that in the absence of PonA2, an alternative mode of peptidoglycan synthesis likely becomes essential. Consistent with this hypothesis, a negative genetic selection identified the l,d-transpeptidase ldtMt2 as essential in the absence of ponA2. The mechanism of ß-lactam resistance we outline is consistent with emerging models of ß-lactam killing, while the investigation of ponA2 downstream and synthetic lethal genes sheds light on the mechanism of cell wall biosynthesis and the interaction between conventional PBPs and l,d-transpeptidases.

Performance of BD Max StaphSR for Screening of Methicillin-Resistant Staphylococcus aureus Isolates among a Contemporary and Diverse Collection from 146 Institutions Located in Nine U.S. Census Regions: Prevalence of mecA Dropout Mutants.

This study determined the performance of BD Max StaphSR and the rate of methicillin-resistant Staphylococcus aureus (MRSA) with an unrecognized staphylococcal cassette chromosome mec (SCCmec) right-extremity junction (MREJ) region among 907 methicillin-resistant S. aureus (MRSA) and 900 methicillin-susceptible S. aureus (MSSA) isolates. The rate of mecA/mecC dropout mutants was also evaluated. Only three MRSA isolates (99.7% sensitivity; 904/907) were classified as MSSA by the BD Max StaphSR assay, due to negative results for MREJ. Eight MSSA isolates (99.1% sensitivity; 892/900) were assigned as MRSA. However, six of these MSSA isolates had the mecA gene confirmed by PCR and sequencing (99.8% sensitivity; 898/900). Overall, 7.1% (64/900) of MSSA isolates showed results compatible with a mecA dropout genotype.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.

BACKGROUND: Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. RESULTS: The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. CONCLUSION: There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system.

High salt stress in Bacillus subtilis: involvement of PBP4* as a peptidoglycan hydrolase.

The study was focused on the role of the penicillin binding protein PBP4* of Bacillus subtilis during growth in high salinity rich media. Using pbpE-lacZ fusion, we found that transcription of the pbpE gene is induced in stationary phase and by increased salinity. This increase was also corroborated at the translation level for PBP4* by western blot. Furthermore, we showed that a strain harboring gene disruption in the structural gene (pbpE) for the PBP4* endopeptidase resulted in a salt-sensitive phenotype and increased sensitivity to cell envelope active antibiotics (vancomycin, penicillin and bacitracin). Since the pbpE gene seems to be part of a two-gene operon with racX, a racX::pRV300 mutant was obtained. This mutant behaved like the wild-type strain with respect to high salt. Electron microscopy showed that high salt and mutation of pbpE resulted in cell wall defects. Whole cells or purified peptidoglycan from WT cultures grown in high salt medium showed increased autolysis and susceptibility to mutanolysin. We demonstrate through zymogram analysis that PBP4* has murein hydrolyze activity. All these results support the hypothesis that peptidoglycan is modified in response to high salt and that PBP4* contributes to this modification.

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli.

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated.

Listeria monocytogenes EGD lacking penicillin-binding protein 5 (PBP5) produces a thicker cell wall.

We report on the cloning of the structural gene for penicillin-binding protein 5 (PBP5), lmo2754. We also describe the enzymatic activity of PBP5 and characterize a mutant lacking this activity. Purified PBP5 has dd-carboxypeptidase activity, removing the terminal D-alanine residue from murein pentapeptide side chains. It shows higher activity against low molecular weight monomeric pentapeptide substrates compared to dimeric pentapeptide compound. Similarly, PBP5 preferentially cleaves monomeric pentapeptides present in high-molecular weight murein sacculi. A Listeria monocytogenes mutant lacking functional PBP5 was constructed. Cells of the mutant are viable, showing that the protein is dispensable for growth, but grow slower and have thickened cell walls.