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Using a Galleria mellonella animal model, we compared the virulence of two sequence type 258 (ST258) KPC-producing Klebsiella pneumoniae strains, which were representative of the two clades of this clonal lineage, with that of isogenic colistin-resistant mgrB mutants. With both strains, the mgrB mutants did not exhibit modification in virulence. In the G. mellonella model, the clade 1 strain (capsular type cps-1 [ wzi29 , producing KPC-2]) was significantly more virulent than the clade 2 strain (capsular type cps-2 [ wzi154 , producing KPC-3]).

Various intracellular bacterial symbionts that provide their host with essential nutrients have much-reduced genomes, attributed largely to genomic decay and relaxed selection. To obtain quantitative estimates of the metabolic function of these bacteria, we reconstructed genome- and transcriptome-informed metabolic models of three xylem-feeding insects that bear two bacterial symbionts with complementary metabolic functions: a primary symbiont, Sulcia, that has codiversified with the insects, and a coprimary symbiont of distinct taxonomic origin and with different degrees of genome reduction in each insect species (Hodgkinia in a cicada, Baumannia in a sharpshooter, and Sodalis in a spittlebug). Our simulations reveal extensive bidirectional flux of multiple metabolites between each symbiont and the host, but near-complete metabolic segregation (i.e., near absence of metabolic cross-feeding) between the two symbionts, a likely mode of host control over symbiont metabolism. Genome reduction of the symbionts is associated with an increased number of host metabolic inputs to the symbiont and also reduced metabolic cost to the host. In particular, Sulcia and Hodgkinia with genomes of ≤0.3 Mb are calculated to recycle ∼30 to 80% of host-derived nitrogen to essential amino acids returned to the host, while Baumannia and Sodalis with genomes of ≥0.6 Mb recycle 10 to 15% of host nitrogen. We hypothesize that genome reduction of symbionts may be driven by selection for increased host control and reduced host costs, as well as by the stochastic process of genomic decay and relaxed selection. IMPORTANCE Current understanding of many animal-microbial symbioses involving unculturable bacterial symbionts with much-reduced genomes derives almost entirely from nonquantitative inferences from genome data. To overcome this limitation, we reconstructed multipartner metabolic models that quantify both the metabolic fluxes within and between three xylem-feeding insects and their bacterial symbionts. This revealed near-complete metabolic segregation between cooccurring bacterial symbionts, despite extensive metabolite exchange between each symbiont and the host, suggestive of strict host controls over the metabolism of its symbionts. We extended the model analysis to investigate metabolic costs. The positive relationship between symbiont genome size and the metabolic cost incurred by the host points to fitness benefits to the host of bearing symbionts with small genomes. The multicompartment metabolic models developed here can be applied to other symbioses that are not readily tractable to experimental approaches.

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK , which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB L82R in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB L82R mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

Sequencing of the bla KPC -positive strain Proteus mirabilis AOUC-001 was performed using both the MiSeq and PacBio RS II platforms and yielded a single molecule of 4,272,433 bp, representing the complete chromosome. Genome analysis showed the presence of several acquired resistance determinants, including two copies of bla KPC-2 carried on a fragment of a KPC-producing plasmid previously described in Klebsiella pneumoniae .

The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35 , while Dbv3 positively controls the expression of four monocistronic transcription units ( dbv4 , dbv29 , dbv36 , and dbv37 ) and of six operons ( dbv2-dbv1 , dbv14-dbv8 , dbv17-dbv15 , dbv21-dbv20 , dbv24-dbv28 , and dbv30-dbv35 ). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation. IMPORTANCE This report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

The POM-1 metallo-β-lactamase is a subclass B3 resident enzyme produced by Pseudomonas otitidis , a pathogen causing otic infections. The enzyme was overproduced in Escherichia coli BL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

We report multifocal detection (four different cities in northern Italy) of Proteus mirabilis isolates resistant to both oxyimino- and 7-α-methoxy-cephalosporins and producing a novel acquired AmpC-like β-lactamase. The enzyme, named CMY-16, is a variant of the CMY/LAT lineage, which differs from the closest homologues, CMY-4 and CMY-12, by a single amino acid substitution (A171S or N363S, respectively) and from CMY-2 by two substitutions (A171S and W221R). Expression of the cloned bla CMY-16 gene in Escherichia coli decreased susceptibility to penicillins, cephalosporins, and aztreonam. Tazobactam was more effective than clavulanate at antagonizing the enzyme activity. Genotyping, by random amplification of polymorphic DNA and pulsed-field gel electrophoresis of genomic DNA digested with SfiI, showed that isolates were clonally related to each other, although not identical. The bla CMY-16 gene was not transferable to E. coli by conjugation or transformation. In all isolates, it was chromosomally located and inserted in a conserved genetic environment. PCR mapping experiments revealed that the bla CMY-16 was flanked by IS Ecp1 and the blc gene, similar to other genes of this lineage from plasmids of Salmonella enterica , Klebsiella spp., and E. coli . Overall, these results revealed multifocal spreading of a CMY-16-producing P. mirabilis clone in northern Italy. This finding represents the first report of an acquired AmpC-like β-lactamase in Proteus mirabilis from Italy and underscores the emergence of similar resistance determinants in the European setting.

Exposure of Escherichia coli MG1655 to environmental bacteriophages results in rapid selection for phage-tolerant subpopulations displaying increased biofilm formation. Analysis of one phage-tolerant strain revealed large amounts of the DNA-binding Dps protein in the outer membrane protein and production of fimbria-like structures. In dps and fimA mutant derivatives of MG1655, no selection of phage-tolerant bacteria upon exposure to bacteriophages occurred, suggesting a role for Dps and type I pili in bacteriophage tolerance.

Twelve isolates of Enterobacteriaceae (1 of Klebsiella pneumoniae , 8 of Escherichia coli , 1 of Proteus mirabilis , and 2 of Proteus vulgaris ) classified as extended-spectrum β-lactamase (ESBL) producers according to the ESBL screen flow application of the BD-Phoenix automatic system and for which the cefotaxime MICs were higher than those of ceftazidime were collected between January 2001 and July 2002 at the Laboratory of Clinical Microbiology of the San Matteo University Hospital of Pavia (northern Italy). By PCR and sequencing, a CTX-M-type determinant was detected in six isolates, including three of E. coli (carrying bla CTX-M-1 ), two of P. vulgaris (carrying bla CTX-M-2 ), and one of K. pneumoniae (carrying bla CTX-M-15 ). The three CTX-M-1-producing E. coli isolates were from different wards, and genotyping by pulsed-field gel electrophoresis (PFGE) revealed that they were clonally unrelated to each other. The two CTX-M-2-producing P. vulgaris isolates were from the same ward (although isolated several months apart), and PFGE analysis revealed probable clonal relatedness. The bla CTX-M-1 and bla CTX-M-2 determinants were transferable to E. coli by conjugation, while conjugative transfer of the bla CTX-M-15 determinant from K. pneumoniae was not detectable. Present findings indicate that CTX-M enzymes of various types are present also in Italy and underscore that different CTX-M determinants can be found in a single hospital and can show different dissemination patterns. This is also the first report of CTX-M-2 in P. vulgaris .

Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS 5 -like, IS 1F -like, or IS Kpn14 ), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

The sigma(S) subunit of RNA polymerase, the product of the rpoS gene, controls the expression of genes responding to starvation and cellular stresses. Using gene array technology, we investigated rpoS-dependent expression at the onset of stationary phase in Escherichia coli grown in rich medium. Forty-one genes were expressed at significantly lower levels in an rpoS mutant derived from the MG1655 strain; for 10 of these, we also confirmed rpoS and stationary-phase dependence by reverse transcription-PCR. Only seven genes (dps, osmE, osmY, sodC, rpsV, wrbA, and yahO) had previously been recognized as rpoS dependent. Several newly identified rpoS-dependent genes are involved in the uptake and metabolism of amino acids, sugars, and iron. Indeed, the rpoS mutant strain shows severely impaired growth on some sugars such as fructose and N-acetylglucosamine. The rpoS gene controls the production of indole, which acts as a signal molecule in stationary-phase cells, via regulation of the tnaA-encoded tryptophanase enzyme. Genes involved in protein biosynthesis, encoding the ribosome-associated protein RpsV (sra) and the initiation factor IF-1 (infA), were also induced in an rpoS-dependent fashion. Using primer extension, we determined the promoter sequences of a selection of rpoS-regulated genes representative of different functional classes. Significant fractions of these promoters carry sequence features specific for Esigma(S) recognition of the -10 region, such as cytosines at positions -13 (70%) and -12 (30%) as well as a TG motif located upstream of the -10 region (50%), thus supporting the TGN(0-2)C(C/T)ATA(C/A)T consensus sequence recently proposed for sigma(S).

The effect of cinnabar on pyrite oxidation by mercury-sensitive and mercury-resistant strains of Thiobacillus ferrooxidans was investigated by using percolation columns. Mercury-resistant strains oxidized pyrite in pyrite-cinnabar mixtures (1 and 10%, wt/wt), whereas a mercury-sensitive strain did not. Elemental mercury was produced by the mercury-resistant strains growing in the pyrite-cinnabar mixtures in percolation columns and in flasks containing cinnabar only. Manometric experiments showed that cinnabar had little effect on oxygen uptake of mercury-sensitive or mercury-resistant cells growing on ferrous sulfate, pyrite, or pyrite-ferrous sulfate mixtures. In addition, shake flask leaching experiments showed that cinnabar had little effect on pyrite oxidation at 1% (wt/wt) but inhibited growth of mercury-sensitive and mercury-resistant strains at 10%. Mercury-resistant strains were unable to grow on cinnabar as an energy source.