Endopeptidase-Mediated Beta Lactam Tolerance

In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall (‘autolysins’) are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall- acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate - sphere formation vs. lysis – after treatment with antibiotics that target cell wall synthesis.     

Cell wall changes in nisin-resistant variants of Listeria innocua grown in the presence of high nisin concentrations

Abstract Two nisin-resistant variants of a strain of Listeria innocua were isolated after growth in the presence of 500 and 4000 IU ml⁻¹ of nisin A showed increased cell wall hydrophobicity, resistance to phage attack and three different cell wall-acting antibiotics, as well as to the peptidoglycan hydrolytic enzymes lysozyme and mutanolysin, as compared to the parental strain. Transmission electron microscopy revealed marked thickening of the wall of nisin-resistant cells with an irregular surface. Differences in thickness were lost after cell wall purification and no significant difference in gross wall composition was observed between the parental and resistant variants. Cell wall changes in nisin-resistant listeriae are attributed to abnormal cell wall synthesis and autolysin inhibition, the latter possibly associated with subtle changes in cell wall structures and function.     

Diversity of plant cell wall esterases in thermophilic and thermotolerant fungi

Fourteen thermophilic and thermotolerant fungal strains isolated from composting soils produced plant cell wall-acting esterases in a medium containing corn cobs and oat spelt xylan. The concentrated and dialyzed protein extracts of these fungi were fractionated using isoelectric-focusing, gels sliced and eluted protein in each slice was assayed for esterase activity against p-nitrophenyl acetate. A total of 84 esterases detected on the basis of pI were found to show distinct preferential substrate specificities towards p-nitrophenyl acetate, p-nitrophenyl ferulate and p-nitrophenyl butyrate, and were putatively classified as acetyl esterases and esterases types I and II. None of the esterases were active against p-nitrophenyl myristate. In addition, these esterases were characterized as acid, neutral or alkaline active.     

Screening of benzalkonium chloride resistance in Listeria monocytogenes strains isolated during cold smoked fish production

AIMS: To determine the susceptibility to disinfectants and cross-resistance to antibiotics in Listeria monocytogenes strains isolated from fish products and the fish-processing environment. METHODS AND RESULTS: Minimal inhibitory concentration assessment, using the agar dilution method, showed 108 of 255 L. monocytogenes isolates with low susceptibility to benzalkonium chloride (BC), commonly used in food industries. Most of them are from raw products of farmed fish during processing, while the remaining resistant isolates were mainly from the environment and finished products irrespective of the fish species. Two BC-resistant isolates were resistant to ethidium bromide (EB). The conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid associated. EB accumulation assays demonstrated that the two BC(R) EB(R) isolates used an efflux pump to expel these substrates whereas a different mechanism was probably used by the majority of the strains with BC(R) EB(S) pattern. No cross-resistance was found with antibiotics. CONCLUSIONS: This study highlights the difference in susceptibilities to BC for L. monocytogenes strains isolated from fish-processing plants and in resistance mechanisms to BC developed by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of BC resistant L. monocytogenes strains could contribute to their adaptation and so explained their survival and persistence in the fish-processing environment.     

Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.     

Comparative transcriptome analysis of Listeria monocytogenes strains of the two major lineages reveals differences in virulence, cell wall, and stress response

Listeria monocytogenes is a food-borne, opportunistic, bacterial pathogen causing a wide spectrum of diseases, including meningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, human listeriosis is mostly associated with strains of serovars 4b, 1/2b, and 1/2a. Within the species L. monocytogenes, three phylogenetic lineages are described. Serovar 1/2a belongs to phylogenetic lineage I, while serovars 4b and 1/2b group in phylogenetic lineage II. To explore the role of gene expression in the adaptation of L. monocytogenes strains of these two major lineages to different environments, as well as in virulence, we performed whole-genome expression profiling of six L. monocytogenes isolates of serovars 4b, 1/2b, and 1/2a of distinct origins, using a newly constructed Listeria multigenome DNA array. Comparison of the global gene expression profiles revealed differences among strains. The expression profiles of two strains having distinct 50% lethal doses, as assessed in the mouse model, were further analyzed. Gene ontology term enrichment analysis of the differentially expressed genes identified differences in protein-, nucleic acid-, carbon metabolism-, and virulence-related gene expression. Comparison of the expression profiles of the core genomes of all strains revealed differences between the two lineages with respect to cell wall synthesis, the stress-related sigma B regulon and virulence-related genes. These findings suggest different patterns of interaction with host cells and the environment, key factors for host colonization and survival in the environment.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell

Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin-based motility, such as CSNK2B, Listeria monocytogenes requires specific host factors, such as CSNK1A1 in order to form productive membrane protrusions and spread from cell to cell.     

Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages

Listeria monocytogenes induces apoptosis in vitro and in vivo in a variety of cell types. However, the mechanism of cell death in L. monocytogenes -infected macrophages was initially reported to be distinct from apoptosis. Here, we studied the mechanism of L. monocytogenes -induced cell death using sensitive fluorescent techniques. We found that caspase-1 activation preceded cell death of macrophages infected with L. monocytogenes , using fluorogenic substrates. Caspase-1 activation was diminished after infection with wild-type L. monocytogenes when cells were treated with NH₄Cl, or if they were infected with a listeriolysin mutant that cannot escape from the phagolysosome. Mitochondrial membrane integrity was preserved during the infection. A particular mechanism of cell death, recently termed 'pyroptosis', is associated with infection by intracellular microorganisms, and has an inherent pro-inflammatory character, due to involvement of caspase-1 activation with consequent IL-1β and IL-18 production. Cell death through caspase-1 activation would constitute a defence mechanism of macrophages which induces cell death to eliminate the bacteria's intracytosolic niche and recruits early host's defences through the secretion of inflammatory cytokines.     

Identification of Listeria monocytogenes genes expressed in response to growth at low temperature

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10 degrees C compared to those grown at 37 degrees C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10 degrees C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.     

从临床感染中分离的九株粘质沙雷氏菌

从临床化脓性感染中分离到九株革兰氏阴性产生脂溶性红色素的小杆菌。经详细鉴定(20种生化反应,120种底物利用试验及G+C mol%测定)为粘质沙雷氏菌(Serratia marcescens)。其中两株能利用3,4-二羟基苯甲酸盐为唯一的碳源而生长,证明此试验适用于本属的种型鉴定。9株菌中有7株细菌属于粘质沙雷氏菌A₂型。药敏试验显示青霉素及其它作用于细胞壁的抗生素对这些菌无效而所试验的氨基糖苷类抗生素几乎全有效。红霉紊、链霉素和复方增效磺胺则各株间的敏感性不同;磺胺虽     

The cell wall binding domain of Listeria bacteriophage endolysin PlyP35 recognizes terminal GlcNAc residues in cell wall teichoic acid

The cell wall binding domains (CBD) of bacteriophage endolysins target the enzymes to their substrate in the bacterial peptidoglycan with extraordinary specificity. Despite strong interest in these enzymes as novel antimicrobials, little is known regarding their interaction with the bacterial wall and their binding ligands. We investigated the interaction of Listeria phage endolysin PlyP35 with carbohydrate residues present in the teichoic acid polymers on the peptidoglycan. Biochemical and genetic analyses revealed that CBD of PlyP35 specifically recognizes the N-acetylglucosamine (GlcNAc) residue at position C4 of the polyribitol-phosphate subunits. Binding of CBDP35 could be prevented by removal of wall teichoic acid (WTA) polymers from cell walls, and inhibited by addition of purified WTAs or acetylated saccharides. We show that Listeria monocytogenes genes lmo2549 and lmo2550 are required for decoration of WTAs with GlcNAc. Inactivation of either gene resulted in a lack of GlcNAc glycosylation, and the mutants failed to bind CBDP35. We also report that the GlcNAc-deficient phenotype of L. monocytogenes strain WSLC 1442 is due to a small deletion in lmo2550, resulting in synthesis of a truncated gene product responsible for the glycosylation defect. Complementation with lmo2550 completely restored display of characteristic serovar 1/2 specific WTA and the wild-type phenotype.     

Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays

Listeria monocytogenes is a pathogenic intracellular microorganism whose infection induces pleiotropic biological changes associated with host cell gene expression regulation. Here we define the gene expression profiles of the human promyelocytic THP1 cell line before and after L. monocytogenes infection. Gene expression was measured on a large scale via oligonucleotide microarrays with probe sets corresponding to 6,800 human genes. We assessed and discussed the reproducibility of the hybridization signatures. In addition to oligonucleotide arrays, we also performed the large scale gene expression measurement with two high-density membranes, assaying for 588 and 18,376 human genes, respectively. This work allowed the reproducible identification of 74 up-regulated RNAs and 23 down-regulated RNAs as a consequence of L. monocytogenes infection of THP1. The reliability of these data was reinforced by performing independent infections. Some of these detected RNAs were consistent with previous results, while some newly identified RNAs encode gene products that may play key roles in L. monocytogenes infection. These findings will undoubtedly enhance the understanding of L. monocytogenes molecular physiology and may help identify new therapeutic targets.     

FSL J1-208, a virulent uncommon phylogenetic lineage IV Listeria monocytogenes strain with a small chromosome size and a putative virulence plasmid carrying internalin-like genes

The bacterial genus Listeria contains both saprotrophic and facultative pathogenic species. A small genome size has been suggested to be associated with the loss of pathogenic potential of L. welshimeri and L. seeligeri. In this paper we present data on the genome of L. monocytogenes strain FSL J1-208, a representative of phylogenetic lineage IV. Although this strain was isolated from a clinical case in a caprine host and has no decreased invasiveness in human intestinal epithelial cells, our analyses show that this strain has one of the smallest Listeria chromosomes reported to date (2.78 Mb). The chromosome contains 2,772 protein-coding genes, including well-characterized virulence-associated genes, such as inlA, inlB, and inlC and the full prfA gene cluster. The small genome size is mainly caused by the absence of prophages in the genome of L. monocytogenes FSL J1-208, and further analyses showed that the total size of prophage-related regions is highly correlated to chromosome size in the genus Listeria. L. monocytogenes FSL J1-208 carries a unique type of plasmid of approximately 80 kbp that does not carry genes annotated as being involved in resistance to antibiotics or heavy metals. The accessory genes in this plasmid belong to the internalin family, a family of virulence-associated proteins, and therefore this is the first report of a potential virulence plasmid in the genus Listeria.     

Phenotypic and genotypic evaluation of Listeria monocytogenes strains isolated from fish and fish processing plants

The aim of this study was to perform the phenotypic and genotypic evaluation of Listeria monocytogenes strains isolated from fish and equipment used in fish processing plants. The prevalence of selected gene-encoding virulence factors among L. monocytogenes strains was assessed by multiplex PCR. The genetic (PFGE method) and protein similarities (MALDI-TOF MS technique) of isolates were determined. Their drug resistance (disk-diffusion method and MIC values), serogroup classification (multiplex-PCR), and the ability to co-aggregate with Salmonella enteritidis were also evaluated. Among 37 L. monocytogenes isolates, 36 strains were found, one of which included two genetically identical isolates (PFGE method). In all examined strains, the following genes were found: hlyA, plcB, plcA, inlA, inlB, prfA, iap, and actA. The presence of virulence genes, mpl, and fbpA was confirmed in 32 (88.9%) strains. It was reported that 30 (83.3%) of the strains belonged to serogroup 1/2a-3a. It was also found that the rate of coaggregation with S. enteritidis bacilli was 16.5–36.3%. Among the investigated L. monocytogenes strains, 25 (69.4%) were sensitive to all antibiotics used. Resistance to penicillin was reported most often among strains (n = 6, 16.7%). The assessment of L. monocytogenes virulence level is an important aspect for the protection of public health. It was reported that strains isolated from fish contain genes coding for virulence factors and some of them are antibiotic-resistant. In our study, it was found that strains with a high degree of genetic similarity also showed a high degree of similarity at the level of protein profiles.     

Tolerance of Listeria monocytogenes to cell envelope-acting antimicrobial agents is dependent on SigB

Mutation of sigB impairs the ability of Listeria monocytogenes to grow in sublethal levels, and to survive in lethal concentrations, of the bacteriocins nisin and lacticin 3147 and the antibiotics ampicillin and penicillin G. SigB may therefore represent an attractive target for the development of new control and treatment strategies for this important pathogen.