Presence Of Β-lactam Antibiotics Research Articles

Signal Peptidase-Mediated Cleavage of the Anti-σ Factor RsiP at Site 1 Controls σP Activation and β-Lactam Resistance in Bacillus thuringiensis.

ABSTRACTIn Bacillus thuringiensis, β-lactam antibiotic resistance is controlled by the extracytoplasmic function (ECF) σ factor σP. σP activity is inhibited by the anti-σ factor RsiP. In the presence of β-lactam antibiotics, RsiP is degraded and σP is activated. Previous work found that RsiP degradation requires cleavage of RsiP at site 1 by an unknown protease, followed by cleavage at site 2 by the site 2 protease RasP. The penicillin-binding protein PbpP acts as a sensor for β-lactams. PbpP initiates σP activation and is required for site 1 cleavage of RsiP but is not the site 1 protease. Here, we describe the identification of a signal peptidase, SipP, which cleaves RsiP at a site 1 signal peptidase cleavage site and is required for σP activation. Finally, many B. anthracis strains are sensitive to β-lactams yet encode the σP-RsiP signal transduction system. We identified a naturally occurring mutation in the signal peptidase cleavage site of B. anthracis RsiP that renders it resistant to SipP cleavage. We find that B. anthracis RsiP is not degraded in the presence of β-lactams. Altering the B. anthracis RsiP site 1 cleavage site by a single residue to resemble B. thuringiensis RsiP results in β-lactam-dependent degradation of RsiP. We show that mutation of the B. thuringiensis RsiP cleavage site to resemble the sequence of B. anthracis RsiP blocks degradation by SipP. The change in the cleavage site likely explains many reasons why B. anthracis strains are sensitive to β-lactams.

Inducible Resistance to β-Lactams in Oxacillin-Susceptible mecA1-Positive Staphylococcus sciuri Isolated From Retail Pork.

Most isolated strains of Staphylococcus sciuri contain mecA1, the evolutionary origin of mecA, but are sensitive to β-lactams (OS-MRSS, oxacillin-susceptible mecA1-positive S. sciuri). In order to improve the efficacy of antibiotic treatment, it is important to clarify whether the resistance of OS-MRSS to β-lactams is an inducible phenotype. In this study, three OS-MRSS strains with oxacillin MIC = 1 μg/ml were isolated from 29 retail pork samples. The resistance of OS-MRSS to β-lactams (MIC > 256 μg/ml) was found to be induced by oxacillin, and the induced resistance was observed to remain stable within a certain period of time. Interestingly, the induced β-lactam resistance was not caused by mecA1, heterogeneous resistance, or any genetic mutation, but mainly due to increased wall teichoic acid (WTA) synthesis that thickened the cell wall. The induced strains also showed slower growth rate, as well as decreased adhesion ability and biofilm thickness. These phenotypes were found to be achieved through altered gene expression in associated pathways, such as the citrate cycle and pentose phosphate pathway. The results challenge the traditional antibiotic sensitivity test. In the presence of β-lactam antibiotics, OS-MRSS that was initially sensitive to β-lactams was observed to gradually develop β-lactam resistance in several days. This often-neglected phenomenon in antibiotic sensitivity tests requires further research attention.

Use of Capillary Zone Electrophoresis Coupled to Electrospray Mass Spectrometry for the Detection and Absolute Quantitation of Peptidoglycan-Derived Peptides in Bacterial Cytoplasmic Extracts.

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.

Interactions of Tea-Derived Catechin Gallates with Bacterial Pathogens.

Green tea-derived galloylated catechins have weak direct antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens and are able to phenotypically transform, at moderate concentrations, methicillin-resistant Staphylococcus aureus (MRSA) clonal pathogens from full β-lactam resistance (minimum inhibitory concentration 256–512 mg/L) to complete susceptibility (~1 mg/L). Reversible conversion to susceptibility follows intercalation of these compounds into the bacterial cytoplasmic membrane, eliciting dispersal of the proteins associated with continued cell wall peptidoglycan synthesis in the presence of β-lactam antibiotics. The molecules penetrate deep within the hydrophobic core of the lipid palisade to force a reconfiguration of cytoplasmic membrane architecture. The catechin gallate-induced staphylococcal phenotype is complex, reflecting perturbation of an essential bacterial organelle, and includes prevention and inhibition of biofilm formation, disruption of secretion of virulence-related proteins, dissipation of halotolerance, cell wall thickening and cell aggregation and poor separation of daughter cells during cell division. These features are associated with the reduction of capacity of potential pathogens to cause lethal, difficult-to-treat infections and could, in combination with β-lactam agents that have lost therapeutic efficacy due to the emergence of antibiotic resistance, form the basis of a new approach to the treatment of staphylococcal infections.

Epidemiological and Genetic Overview of the Klebsiella pneumoniae Carbapenemases (KPCs) in K. pneumoniae Isolated from the Clinical Samples in Iran

Background: The prevalence of carbapenem-resistant Enterobacteriaceae, especially Klebsiella pneumoniae carbapenemase (KPC), has been recently reported worldwide. Therefore, there is an indispensable need for precise and rapid detection of these carbapenemases. Objectives: This study aimed to propose an accurate and rapid method for detecting K. pneumoniae carbapenemase genes from clinical samples, using reverse transcription-polymerase chain reaction (RT-PCR) and to evaluate the expression of these genes in the presence of β-lactam antibiotic by real-time PCR assay.Methods: One hundred and eighty-one K. pneumoniae strains were collected from patients presenting to Firoozgar Hospital of Tehran, Iran. The strains were tested using the disk diffusion method, the modified Hodge test (MHT), and E-test minimum inhibitory concentration (MIC). Next, reverse transcription-PCR method was applied for the identification of bla OXA-23 and bla OXA-48 genes. Finally, expression of genes was measured by real-time PCR assay in the presence and absence of β-lactam antibiotic.Results: Phenotypic testing also showed a high level of antibiotic resistance, while the genotypic methods indicated the presence and expression of carbapenemase genes.Conclusions: The findings suggest revisions in the current antibiotic therapy protocols, considering the high expression level of resistant carbapenemases to K. pneumoniae strains.

GENE EXPRESSION of blaOXA-51-like and blaOXA-23 in RESPONSE to β-Lactam ANTIBIOTIC IN CLINICALLY ISOLATED ACINETOBACTER BAUMANNII AND ACINETOBACTER LOWFFII FROM URINE SAMPLES

In this study, two isolates A. lowffii, and A. baumannii were obtained clinically, and genomically identified by 16S rRNA assessment with the accession number (MH685113.1 and MH685112.1) for A. lowffii and A. baumannii respectively. The sensitivity profile of the isolates was variable, and A. baumannii was the most resistant strain towards a wide range of antibiotics, and it did not show any growth defect in the presence of β-lactam antibiotic, in comparison with A. lowffii. We identified that the blaOXA-23 gene was responsible for imipenem resistance in A. baumannii, whereas, blaOXA-51-like was moderately confer resistance towards A. lowffii which lack of blaOXA-23. This was determined when the isolates were subjected to qRT-PCR. We identified that the blaOXA-23 gene was increased about 1-fold in the presence of imipenem, whereas, blaOXA-51-like did not increased in comparison to the control. Bioinformatic analyses revealed that blaOXA-23 is located in the cytoplasm, and blaOXA-like 51 is located in the periplasm. Our hypothesis suggests that blaOXA-23 gene have a major contribution in the outbreak of multidrug resistance Acinetobacter species.

BPEI-Induced Delocalization of PBP4 Potentiates β-Lactams against MRSA.

With its high morbidity rate and increasing resistance to treatment, methicillin-resistant Staphylococcus aureus (MRSA) is a grave concern in the medical field. In methicillin-susceptible strains, β-lactam antibiotics disable the penicillin binding proteins (PBPs) that cross-link the bacterial cell wall. However, methicillin-resistant strains have PBP2a and PBP4, which continue enzymatic activity in the presence of β-lactam antibiotics. The activity of PBP2a and PBP4 is linked to the presence of wall teichoic acid (WTA); thus, WTA has emerged as a target for antibiotic drug discovery. In this work, we disable WTA in situ using its anionic phosphodiester backbone to attract cationic branched polyethylenimine (BPEI). Data show that BPEI removes β-lactam resistance in common MRSA strains and clinical isolates. Fluorescence microscopy was used to investigate this mechanism of action. The results indicate that BPEI prevents the localization of PBP4 to the cell division septum, thereby changing the cellular morphology and inhibiting cell division. Although PBP4 is not required for septum formation, proper cell division and morphology require WTA; BPEI prevents this essential function. The combination of BPEI and β-lactams is bactericidal and synergistic. Because BPEI allows us to study the role of WTA in the cell wall without genetic mutation or altered translocation of biomolecules and/or their precursors, this approach can help revise existing paradigms regarding the role of WTA in prokaryotic biochemistry at every growth stage.

Cell Shape and Antibiotic Resistance Are Maintained by the Activity of Multiple FtsW and RodA Enzymes in Listeria monocytogenes.

Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that the Bacillus subtilis RodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division-specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen Listeria monocytogenes carries genes that encode up to six FtsW/RodA homologs; however, their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly, L. monocytogenes carries a gene that encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2, and RodA3, and their combined absence is lethal. Cells of a rodA1 rodA3 double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a rodA3 mutant was more susceptible to the β-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights that L. monocytogenes genes encode a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division, and antibiotic resistance.IMPORTANCE The human pathogen Listeria monocytogenes is usually treated with high doses of β-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically on L. monocytogenes, and the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form of Listeria infection, which can be fatal in up to 30% of cases. The development of new strategies to treat Listeria infections is necessary. Here we show that the expression of some of the FtsW and RodA enzymes of L. monocytogenes is induced by the presence of β-lactam antibiotics, and the combined absence of these enzymes makes bacteria more susceptible to this class of antibiotics. The development of antimicrobial agents that inhibit the activity or production of FtsW and RodA enzymes might therefore help to improve the treatment of Listeria infections and thereby lead to a reduction in mortality.

An active β-lactamase is a part of an orchestrated cell wall stress resistance network of Bacillus subtilis and related rhizosphere species.

A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d-leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the β-lactamase PenP. This β-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to β-lactam resistance under different conditions. To test whether β-lactam resistance and β-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five β-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental β-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of β-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between β-lactams and β-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils.

Can β-Lactam Antibiotics Be Resurrected to Combat MRSA?

The use of β-lactam antibiotics to treat infections caused by Staphylococcus aureus has been severely compromised by the acquisition by horizontal gene transfer of a gene that encodes the β-lactam-insensitive penicillin-binding protein PBP2a. This allows methicillin-resistant S. aureus (MRSA) to proliferate in the presence of β-lactam antibiotics. Paradoxically the dependence on PBP2a for the essential transpeptidase activity in cell wall peptidoglycan biosynthesis is the 'Achilles heel' of MRSA. Compounds that disrupt the divisome, wall teichoic acid, and functional membrane microdomains act synergistically with β-lactams against MRSA. These include drugs such as statins that are widely used in human medicine. The antibiotics vancomycin and daptomycin are also synergistic with β-lactams, and combinations have been employed to treat persistent MRSA infections. An additional benefit of exposing MRSA to β-lactams could be a reduction in virulence mediated by interfering with the global regulator Agr. The mechanistic basis of synergy is discussed, and the possibility that β-lactams can be resurrected to combat MRSA infections is explored.

Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli.

Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs) that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC–lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of β-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN–lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σE. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics.

Method Based on theβ-Lactamase PenPC Fluorescent Labeled forβ-Lactam Antibiotic Quantification in Human Plasma

Recently, Wong et al. have successfully developed a fluorescent biosensor based on the PenPC β-lactamase which changes its intrinsic fluorescence in presence of β-lactam antibiotics (BLAs). Here, we studied systematically this correlation among the fluorescence change of the biosensor and the concentration of different BLAs aimed at developing a novel method for estimating the concentration of a wide range of BLAs. This method showed high precision and specificity and very low interference from clinically relevant samples. We were able to monitor the pharmacokinetics of meropenem in healthy volunteers as well as in an ill animal model too, indicating that the implemented method could be suitable for clinical practice.

Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam-Daptomycin Synergy.

The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP-β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response.

Indirect resistance to several classes of antibiotics in cocultures with resistant bacteria expressing antibiotic-modifying or -degrading enzymes.

Indirect resistance (IR), the ability of an antibiotic-resistant population of bacteria to protect a susceptible population, has been previously observed for β-lactamase-producing bacteria and associated with antimicrobial treatment failures. Here, we determined whether other resistance determinants could cause IR in the presence of five other classes of antibiotics. A test was designed to detect IR and 14 antibiotic resistance genes were tested in the presence of 13 antibiotics from six classes. A bioassay was used to measure the ability of resistance-causing enzymes to decrease the concentration of active antibiotics in the medium. We confirmed IR in the presence of β-lactam antibiotics (ampicillin and mecillinam) when TEM-1A was expressed. We found that bacteria expressing antibiotic-modifying or -degrading enzymes Ere(A), Tet(X2) or CatA1 caused IR in the presence of macrolides (erythromycin and clarithromycin), tetracyclines (tetracycline and tigecycline) and chloramphenicol, respectively. IR was not observed with resistance determinants that did not modify or destroy antibiotics or with enzymes modifying aminoglycosides or degrading fosfomycin. IR was dependent on the resistance enzymes decreasing the concentration of active antibiotics in the medium, hence allowing nearby susceptible bacteria to resume growth once the antibiotic concentration fell below their MIC. IR was not limited to β-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy.

Answer to September 2015 Photo Quiz

Answer: Community-acquired pneumonia caused by Acinetobacter baumannii. The Gram-positive rods seen on the initial Gram stain from the blood culture broth were later identified as Acinetobacter baumannii. A. baumannii is a strictly aerobic, nonfermenting, oxidase-negative, catalase-positive coccus/coccobacillus that grows well on both blood agar and MacConkey agar. In Gram-stained preparations, it typically appears as Gram-negative diplococci with an arrangement resembling that of Neisseria spp. (1). It is well recognized that some Acinetobacter isolates tend to retain crystal violet and resist decolorization (2) and may initially appear as Gram-positive cocci in direct smears prepared from positive blood culture bottles (3). This may explain the Gram-positive appearance of the original Gram-stained smear. Of note, there was a similar report of A. baumannii presenting as Gram-positive bacilli in a bronchial washing specimen from a patient with fulminant A. baumannii community-acquired pneumonia (4). However, the mechanism by which this phenomenon occurs remains unclear. The unusual bacillary appearance may be contributed to by the growth phase of the organism as well as the presence of antibiotic in the blood. It was reported that Acinetobacter spp. are plump rods measuring 0.9 to 1.6 by 1.5 to 2.5 μm during exponential growth and become coccoid after entry into the stationary phase (2). The organisms seen on the original Gram stain, however, measured up to 6 μm in length. This morphological change is likely caused by the presence of sub-MIC levels of β-lactam antibiotic in the blood, amoxicillin in this case. This effect has been described to occur in various Gram-negative bacilli in the presence of β-lactam antibiotics, which were proposed to inhibit septum formation, resulting in the elongated morphology (5, 6). The subsequent Gram-stained smear of the isolate from sheep blood agar showed Gram-negative diplococci, in contrast to the Gram-positive bacilli seen in the original smear. The isolate was identified as A. baumannii by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Bruker). The Vitek 2 system (GN ID card) and an API 20 NE strip identified the organism as a member of the Acinetobacter baumannii-Acinetobacter calcoaceticus complex. A. baumannii is ubiquitous in freshwater and soil. It is an opportunistic pathogen that plays a significant role in nosocomial infections and hospital outbreaks. It is also an uncommon cause of severe community-acquired pneumonia. While health care-associated A. baumannii is often multidrug resistant, strains of A. baumannii causing community-acquired pneumonia are usually susceptible to multiple antibiotics. The strain in this case tested susceptible to ampicillin-sulbactam, piperacillin-tazobactam, cefepime, gentamicin, ciprofloxacin, and imipenem via disk diffusion testing. The patient was empirically treated with intravenous amoxicillin-clavulanate and oral clarithromycin, which were subsequently changed to ampicillin-sulbactam on the basis of susceptibility testing. However, on the third day of admission, he developed a fever, and a repeat chest X-ray showed progression of consolidation. Repeat cultures were negative, and he was treated with piperacillin-tazobactam. He completed 10 days of antibiotics in total and returned home in a stable condition. This case illustrates that A. baumannii can present with unusual Gram stain findings in clinical specimens. Both laboratory and clinical personnel should be aware that in the appropriate clinical settings, A. baumannii infection should be considered a differential diagnosis despite seemingly incongruent Gram stain findings. (See page 2797 in this issue [doi:10.1128/JCM.02551-13] for photo quiz case presentation.)

Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

Extended spectrum β-lactamase-producing Escherichia coli forms filaments as an initial response to cefotaxime treatment.

Backgroundβ-lactams target the peptidoglycan layer in the bacterial cell wall and most β-lactam antibiotics cause filamentation in susceptible Gram-negative bacteria at low concentrations. The objective was to determine the initial morphological response of cephalosporin resistant CTX-M-1-producing E. coli to cefotaxime and to determine whether the response depended on the growth phase of the bacterium and the concentration of antibiotic.ResultsTwo antibiotic resistant strains carrying blaCTX-M-1 on the chromosome and on an IncI1 plasmid and three sensitive strains were used in this study. The resistant strains displayed elongated cells when exposed to cefotaxime at sub-inhibitory as well as therapeutic concentrations (1 to 512 mg/L of cefotaxime) in both lag and early exponential phase, suggesting that the elongation was an initial response mechanism to the antibiotic. Normal sized cells were the dominant cell type in exponential and stationary growth phase. No elongated cells were seen in cultures without cefotaxime. In cultures with high concentrations of cefotaxime (128–512 mg/L), no growth other than initial filamentation was observed, but spheroplats appeared after 14–17 hours in cultures of the resistant strains. Filaments were also observed in sensitive control strains with sub-inhibitory concentrations of cefotaxime.ConclusionsWe showed that E. coli resistant to β-lactams by an extended-spectrum β-lactamase, blaCTX-M-1, produced filaments when exposed to cefotaxime. The filament formation was restricted to early growth phases and the time the cells grew as filaments was antibiotic concentration dependent. This indicates that antibiotic resistant E. coli undergo the same morphological changes as sensitive bacteria in the presence of β-lactam antibiotic. It was showed that the filament formation was an initial response to the antibiotics.

Gene and Protein Network Analysis of AmpC β Lactamase.

AmpC β-lactamase is a cephalosporinase, which exhibits resistance against all existing β-lactam antibiotics except carbapenems. Their occurrence in many bacterial pathogens poses a threat to public health and is a growing concern in the medical world. The ampC gene is highly inducible in the presence of β-lactam antibiotics and can be expressed in high levels due to mutation. This inducible expression is regulated by several functional genes. Several studies on functional relationship of these genes and its resistance mechanisms are carried out but it still lacks comprehensible evidences. Thus, in our current study, we used computational gene networks to analyze ampC gene. Based on its interaction type, co-expression, Gene Ontology, and text mining, a functional interaction network is constructed. Around 247 functional genes in 15 different bacterial genus have a functional association with ampC gene. It is predicted that 19.8% ampD, 13.3% frdD, 8.5% gcvA, 2.4% ampR, and 55.7% of other functional partners are associated with ampC gene. Our present study provides a glimpse about the functional gene network of ampC gene and also provides the integrated evidence for ampC gene in regulating the β-lactamase production and its role in antibiotic resistance.

Extended-spectrum β-lactamase and carbapenemase-producing Aeromonas species in wild animals from Portugal

Aeromonas are Gram-negative, facultative-anaerobic, non-spore-forming, glucose-fermenting, oxidase- and catalase-positive rods (Martin-Carnahan and Joseph 2005). Apart from fish, which are widely reported hosts for Aeromonads, insects, crustaceans, reptiles, birds and mammals...

Exposure of Clinical MRSA Heterogeneous Strains to β-Lactams Redirects Metabolism to Optimize Energy Production through the TCA Cycle

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤0.1%) of the population expresses resistance to oxacillin (OXA) ≥10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.