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Introduction: Clinicians commonly escalate empiric antibiotic therapy due to poor clinical progress without microbiology guidance. When escalating, they should take account of how resistance to an initial antibiotic affects the probability of resistance to subsequent options. The term "escalation antibiogram" (EA) has been coined to describe this concept. One difficulty when applying the EA concept to clinical practice is understanding the uncertainty in results and how this changes for specific patient subgroups. Methods: A Bayesian model was developed to estimate antibiotic resistance rates in Gram-negative bloodstream infections based on phenotypic resistance data. The model generates a series of “credible” curves to fit the resistance data, each with the same probability of representing the true rate given the inherent uncertainty. To avoid overfitting, an integrated penalisation term adaptively smooths the curves given the level of evidence. Results: Rates of resistance to empiric first-choice and potential escalation antibiotics were calculated for the whole hospitalised population based on 10,486 individual bloodstream infections, and for a range of specific patient groups, including ICU (intensive care unit), haematolo-oncology, and paediatric patients. The model generated an expected value (posterior mean) with 95% credible interval to illustrate uncertainty, based on the size of the patient subgroup. For example, the posterior means of piperacillin/tazobactam resistance rates in Gram-negative bloodstream infection are different between patients on ICU and the general hospital population: 27.3% (95% CI 18.1–37.2 vs. 13.4% 95% CI 11.0–16.1) respectively. The model can also estimate the probability of inferiority between two antibiotics for a specific patient population. Differences in optimal escalation antibiotic options between specific patient groups were noted. Conclusions: EA analysis informed by our Bayesian model is a useful tool to support empiric antibiotic switches, providing an estimate of local resistance rates, and a comparison of antibiotic options with a measure of the uncertainty in the data. We demonstrate that EAs calculated for the whole hospital population cannot be assumed to apply to specific patient group.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes that hydrolyze β-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50values of 0.3–7.2 μM; Kivalues of 0.03–1.5 μM). Minimum inhibitory concentration assays demonstrated potentiation of β-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N–C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

ABSTRACTAcetamido derivatives of the naturally antibacterial non-β-lactam lactivicin (LTV) have improved activity against their penicillin binding protein targets and reduced hydrolysis by β-lactamases, but penetration into Gram-negative bacteria is still relatively poor. Here we report that modification of the LTV lactone with a catechol-type siderophore increases potency 1,000-fold against Stenotrophomonas maltophilia, a species renowned for its insusceptibility to antimicrobials. The MIC90of modified lactone compound 17 (LTV17) against a global collection of extensively drug-resistant clinical S. maltophiliaisolates was 0.063 μg · ml−1. Sideromimic modification does not reduce the ability of LTVs to induce production of the L1 and L2 β-lactamases in S. maltophiliaand does not reduce the rate at which LTVs are hydrolyzed by L1 or L2. We conclude, therefore, that lactivicin modification with a siderophore known to be preferentially used by S. maltophiliasubstantially increases penetration via siderophore uptake. LTV17 has the potential to be developed as a novel antimicrobial for treatment of infections by S. maltophilia. More generally, our work shows that sideromimic modification in a species-targeted manner might prove useful for the development of narrow-spectrum antimicrobials that have reduced collateral effects.

ABSTRACTIt has been reported that targeted disruption of ampDI or mrcAcauses β-lactamase hyperproduction in Stenotrophomonas maltophilia. We show here that β-lactamase-hyperproducing laboratory selected mutants and clinical isolates can have wild-type ampDI and mrcAgenes, implicating mutation of at least one additional gene in this phenotype. The involvement of mutations at multiple loci in the activation of β-lactamase production in S. maltophiliareveals that there are significant deviations from the enterobacterial paradigm of AmpR-mediated control of β-lactamase induction. We do show, however, that S. maltophiliaampDI can complement a mutation in Escherichia coliampD. This suggests that an anhydromuropeptide degradation product of peptidoglycan is used to activate AmpR in S. maltophilia, as is also the case in enteric bacteria.

ABSTRACTA divergently oriented ampRhas been located upstream of blaL2in Stenotrophomonas maltophilia. AmpR is necessary for L1 and L2 β-lactamase induction in response to β-lactam challenge, and activation of AmpR is sufficient to induce L1 and L2 production. L1 induction requires more activation of AmpR than does L2 induction.

Objectives: To test whether smeDEF overexpression leads to a predictable multi-drug resistance phenotype in Stenotrophomonas maltophilia and to measure the frequency with which smeDEF overexpression occurs in clinical isolates and in spontaneous drug-resistant mutants.Methods: Overexpression of smeDEF was induced in clinical isolates by the introduction of chromosomal mutations in smeT using a gene-replacement approach. Spontaneous drug-resistant mutants were selected using greater than MIC concentrations of various antimicrobial agents. Levels of smeE and smeF mRNAs were quantified using RT–PCR; MICs were determined using Etest.Results: Of 20 spontaneous S. maltophilia drug-resistant mutants tested, four overexpressed smeDEF, but only two carried mutations within smeT. Of 30 clinical isolates tested, 6 significantly overexpressed smeDEF. One of these had an IS1246-like element embedded within the putative SmeT binding site in the smeDEF promoter. All smeDEF overexpressing derivatives of an isolate had the same resistance profile; derivatives that did not overexpress smeDEF did not share this resistance profile. However, no consistent phenotype could be associated with smeDEF overexpression in S. maltophilia isolates per se.Conclusions: SmeT is not the only gene product that affects smeDEF expression. IS element insertion is a viable mechanism by which smeDEF expression can be derepressed. There is evidence for a background-specific, predictable effect on resistance profile when smeDEF is overexpressed, but the variability of backgrounds encountered means no general SmeDEF-mediated phenotype can be defined. There is strong evidence for the existence of as yet unidentified multi-drug efflux pumps in this species.

Aims: To test the hypothesis that Stenotrophomonas maltophilia isolates from certain phylogenetic groups have predictable β-lactamase expression and β-lactam resistance profiles.Methods: Isolates were grouped using sequences of the 16S rRNA gene and smeT–smeD intergenic region. β-Lactamase activities in cell extracts were quantified spectrophotometrically and β-lactam MICs were determined using agar dilution methodology and Etest as appropriate.Results: A collection of 50 clinical S. maltophilia isolates from Europe and North, South and Central America were phylogenetically grouped. Group ‘A’ (22 out of 50) includes remarkably genetically homogeneous isolates; group ‘B’ (17 out of 50) includes isolates that are genetically heterogeneous and quite distinct from those of group A. Members of these two groups are, however, indistinguishable in terms of their β-lactam resistance and β-lactamase expression phenotypes. In contrast, isolates from group ‘C’, which are less common (8 out of 50), are considerably more susceptible to β-lactams owing to reduced inducibility of β-lactamase expression following β-lactam challenge.Conclusions: The majority of S. maltophilia clinical isolates behave similarly in terms of β-lactamase expression and β-lactam resistance properties, despite considerable phylogenetic variability.

Bulgecin A, a sulphonated N-acetyl-D-glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a β-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-β-lactamases. Using steady-state kinetic analysis with nitrocefin as the β-lactam substrate, bulgecin A competitively inhibited the metallo-β-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-β-lactamase from Aeromonas veronii bv. sobria, ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-β-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-β-lactamases via the terminal sulphonate group on the sugar moiety.

Objectives: To determine the most likely evolutionary pathway that has led to the development of extended-spectrum SHV derivatives, and to the mobilization of blaSHV. Materials and methods: Evolutionary mapping used a shortest-path analysis of aligned blaSHV variants, and other basic bioinformatic approaches, such as CLUSTAL W and Blast were employed. Results: Two main branches of the blaSHV evolutionary tree were located; both are derived from variant blaSHV-1 alleles. Identical mutations, responsible for extended-spectrum SHV substrate profiles, have been selected independently in each branch. There is evidence for a pool of non-mobile blaSHV framework sequences. Analysis of the genome sequence of Klebsiella pneumoniae confirms the chromosomal origin of blaSHV, whose mobilization has occurred at least twice, once for each of the main evolutionary branches. Both these mobilization events have been catalysed by IS26. Evolution of blaSHV to give common extended-spectrum variants is most likely to have occurred following mobilization. Conclusions: These data shed new light on the evolution and mobilization of blaSHV, and these observations may be useful in predicting what might happen in future, both for blaSHV, and for other β-lactamase genes.

Legionella pneumophila Philadelphia-1 (ATCC 33152) produces a serine active site beta-lactamase. The chromosomal gene that encodes this enzyme, loxA, has been cloned by PCR using information from the L. pneumophila Philadelphia-1 genome sequencing project. LoxA is a class 2d penicillinase, and its sequence puts it into the molecular class D beta-lactamase family, although phylogenetic analysis shows that LoxA forms a distinct branch in the OXA family along with the LoxA homologue, OXA-29, from Legionella gormanii ATCC 33297(T). Upstream of loxA on the L. pneumophila Philadelphia-1 chromosome is a two-gene locus similar to that found linked to the beta-lactamase genes of Gram-positive bacteria. The unit consists of loxI, encoding a homologue of the Gram-positive beta-lactamase expression regulator, and pbpX, encoding a putative penicillin-binding transpeptidase. Despite the presence of beta-lactamase regulator homologues, we could find no evidence of LoxA induction upon challenge of L. pneumophila Philadelphia-1 with beta-lactams.

Glycopeptides, such as vancomycin, are frequently the antibiotics of choice for treatment of infections caused by the now common methicillin-resistant Staphylococcus aureus (MRSA). Incidences of vancomycin resistance in S. aureus (VRSA) have been increasing worldwide for the last 5 years. Complex mechanisms producing changes in cell wall content and composition generate the VRSA phenotype, but the genetic basis of these changes has not yet been determined. To facilitate the genetic investigation, entire genome sequences of the archetypal VRSA (Mu50), and vancomycin-susceptible MRSA strains N315, EMRSA 16 and COL were compared. The in silico analysis revealed several loss-of-function mutations in Mu50, affecting important cell wall biosynthesis and intermediary metabolism genes, not previously reported. The new findings provide further evidence for the hypothesis that vancomycin resistance in Mu50 is due to fundamental changes, important to metabolic pathways that impinge on peptidoglycan biosynthesis. These observations will inform targeted experiments aimed at a complete understanding of the mechanism(s) of vancomycin resistance in S. aureus Mu50 and other VRSA strains.

Caulobacter crescentus101123 possesses a gene (Mbl1b) encoding a metallo‐β‐lactamase with 32% amino acid identity to the L1 metallo‐β‐lactamase from Stenotrophomonas maltophilia. The gene was cloned into an expression vector and the enzyme, Mbl1b, was expressed in Escherichia coli. Mbl1b was purified. Catalytic properties for several antibiotics were determined. The enzyme exhibits Michaelis–Menten kinetics for imipenem, meropenem and nitrocefin but substrate inhibition kinetics with cefoxitin, cefaloridine, penicillin G and ampicillin. A homology model predicts Mbl1b has the same structural fold as other metallo‐β‐lactamases with a detailed structure very similar to L1 but whereas L1 is a homotetramer, Mbl1b is monomeric. The main differences between Mbl1 and L1 are in the N‐terminal region.

We have identified nine genes, the expression of which are regulated by the CreBC two-component system: the first members of the cre regulon. They are divided into eight transcriptional units, each having a promoter-proximal TTCACnnnnnnTTCAC “cre-tag” motif. The cre regulon genes are: theackA/ptaoperon, the products of which collectively catalyze the conversion of acetyl-CoA into acetate and ATP; talA, which encodes an enzyme involved in the mobilization of glyceraldehyde-3-phosphate into the pentose phosphate pathway; radC, which encodes a RecG-like DNA recombination/repair function; malE, which is the first gene in the malEFGmaltose transporter operon;trgB, which encodes an ADP-ribose pyrophosphorylase; and three other genes, creD, yidSandyieI, the products of which have not been assigned a function. Expression of each of these cre regulon genes is induced via CreBC during growth in minimal media, with the exception ofmalE, which is more tightly repressed. The diverse functions encoded by the cre regulon suggest that CreBC is a global regulator that sits right at the heart of metabolic control inEscherichia coli.

A ColE2-like, cryptic plasmid, pUB6060, of 5.8 kb has been found in a clinical isolate of Plesiomonas shigelloides. The complete sequence of pUB6060 has been determined and reveals a number of interesting features about the plasmid. The ColE2-like replication locus is linked to a functional ColE1-like mobilization locus. Replication is, unusually for ColE2 replicons, DNA polymerase-I-independent and may involve two, rather than the usual one, plasmid-encoded functions. Additionally, it carries two ORFs encoding products of unknown function. The pUB6060 replicon maintains a moderate plasmid copy number (10 per chromosome copy) and permits replication in diverse gram-negative bacteria.

ABSTRACTAn approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 β-lactamase genes. The location of L1andL2on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes,L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1(L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lowerkcat/Kmratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, andL2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275L2(L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.

A constitutively expressed β-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of blaTEM2 (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The blaTEM gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize blaTEM on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM β-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile β-lactamase genes in the hospital environment.

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240–10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.

There is mounting evidence that in fat and other insulin-sensitive cells activation of protein synthesis may involve the dissociation of a protein (4E-BP1) from eukaryotic initiation factor (eIF)-4E thus allowing formation of the eIF-4F complex. This study compares the effects of insulin and epidermal growth factor (EGF) on the phosphorylation of 4E-BP1 in fat-cells (followed by gel-shift assays and incorporation of 32P) and on its association with eIF-4E. Several lines of evidence suggest that mitogen-activated protein kinase (MAP kinase) is not involved in these effects of insulin. Insulin causes much more extensive phosphorylation and dissociation of 4E-BP1 from eIF-4E than EGF, although EGF activates MAP kinase to a much greater extent than insulin. Moreover, MAP kinase does not phosphorylate 4E-BP1 when it is complexed with eIF-4E. In contrast, insulin activates the 40S ribosomal protein S6 kinase (p70S6K) 18-fold compared with a 2-fold activation by EGF, and the time course of this activation is similar to the phosphorylation and dissociation of 4E-BP1. Rapamycin, a specific inhibitor of the activation of this latter kinase, inhibits dissociation of 4E-BP1 from eIF-4E in cells incubated with insulin but reveals a phosphorylated form of 4E-BP1 which remains bound to eIF-4E. It is concluded that in rat epididymal fat-cells, the effects of insulin on 4E-BP1 involves multiple phosphorylation events. One phosphorylation event is rapamycin-insensitive, occurs only on bound 4E-BP1 and does not initiate dissociation. The second event does result in dissociation and is blocked by rapamycin, suggesting that the p70S6K signalling pathway is involved: p70S6K itself is probably not involved directly as this kinase does not phosphorylate 4E-BP1 in vitro.

Meropenem is a clinically important antibacterial reserved for treatment of multiresistant infections. In meropenem-resistant bacteria of the family Enterobacterales, New Delhi metallo-β-lactamase 1 (NDM-1) is considerably more common than IMP-1, despite both metallo-β-lactamases (MBLs) hydrolyzing meropenem with almost identical kinetics.

ABSTRACTA Stenotrophomonas maltophiliamutant that coordinately hyper-expresses three resistance nodulation division-type efflux pump genes, smeZ, smeJ, and smeK, has been identified. SmeZ is responsible for elevating aminoglycoside MICs; SmeJ and SmeK are jointly responsible for elevating tetracycline, minocycline, and ciprofloxacin MICs and conferring levofloxacin resistance. One clinical isolate with this same phenotype was identified from a sample of six, and the isolate also coordinately hyper-expresses smeZand smeJK, confirming the clinical relevance of our findings.

Fluoroquinolone resistance in Stenotrophomonas maltophiliais multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622in S. maltophiliaK279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS).

Serine β-lactamases are dominant causes of β-lactam resistance in Klebsiella pneumoniaeisolates. Recently, this has driven clinical deployment of the β-lactam–β-lactamase inhibitor pairs ceftazidime/avibactam and meropenem/vaborbactam. We show that four steps, i.e., ompK36and ramRmutation plus carriage of OXA-232 and KPC-3-D178Y variant β-lactamases, confer ceftazidime/avibactam and meropenem/vaborbactam resistance when both pairs are used together.

Colistin resistance in Klebsiella pneumoniaeis predominantly caused by mutations that increase expression of the arn(also known as pbgor pmrF) operon. Expression is activated by the PhoPQ and PmrAB two-component systems. Constitutive PhoPQ activation occurs directly by mutation or following loss of MgrB. PhoPQ may also cross-activate PmrAB via the linker protein PmrD. Using proteomics, we show that MgrB loss causes a wider proteomic effect than direct PhoPQ activation, suggesting additional targets for MgrB.

Meropenem-vaborbactam resistance in Klebsiella pneumoniaeisolates is associated with loss-of-function mutations in the OmpK35 and OmpK36 porins. We identify two previously unknown loss-of-function mutations that confer cefuroxime resistance in K. pneumoniaeisolates. The proteins lost were NlpD and KvrA; the latter is a transcriptional repressor that controls capsule production. We demonstrate that KvrA loss reduces OmpK35 and OmpK36 porin production, which confers reduced susceptibility to meropenem-vaborbactam in a KPC-3-producing K. pneumoniaeisolate.

It has been reported that Stenotrophomonas maltophilia L1 and L2 beta-lactamase expression is coordinated. We have isolated S. maltophilia mutants where (i) L1 is constitutively hyper-expressed and L2 is inducible; (ii) L2 is hyper-expressed and L1 is inducible; and (iii) L1 and L2 are constitutively hyper-expressed. The frequency of isolating type 1 and 2 mutants is c. 10(-7), indicating that promoter mutations are probably not involved and providing strong evidence that L1 and L2 expression is not, after all, coordinated. The frequency of isolating type 3 mutants is c. 10(-9), however, implying that there is a significant overlap between the regulatory mechanisms.

Aminoglycoside resistance in Stenotrophomonas maltophiliais multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory-selected mutants and clinical isolates, we show here that damage to the 50S ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ.

Mutants with enhanced growth in the presence of an antibiotic are more difficult to identify than mutants where the antibiotic’s MIC increases, because they are not amenable to lethal selection in vitro. We report that activatory mutations in the CreC signal sensor enhance growth of Escherichia coliin the presence of cefoxitin, cefotaxime, and meropenem, without increasing their MICs. Enhanced growth is dependent on overproduction of the inner membrane creregulon protein CreD.

ABSTRACTThe hyperproduction of chromosomally encoded β-lactamases is a key method of acquired resistance to ceftazidime, aztreonam, and, when seen in backgrounds having reduced envelope permeability, carbapenems. Here, we show that the loss of Mpl, a UDP-muramic acid/peptide ligase, is a common and previously overlooked cause of chromosomally encoded β-lactamase hyperproduction in clinical isolates of Stenotrophomonas maltophiliaand Pseudomonas aeruginosa, important pathogens notorious for their β-lactam-resistant phenotypes.

ABSTRACTFluoroquinolone resistance in Gram-negative bacteria is multifactorial, involving target site mutations, reductions in fluoroquinolone entry due to reduced porin production, increased fluoroquinolone efflux, enzymes that modify fluoroquinolones, and Qnr, a DNA mimic that protects the drug target from fluoroquinolone binding. Here we report a comprehensive analysis, using transformation and in vitromutant selection, of the relative importance of each of these mechanisms for fluoroquinolone nonsusceptibility using Klebsiella pneumoniaeas a model system. Our improved biological understanding was then used to generate 47 rules that can predict fluoroquinolone susceptibility in K. pneumoniaeclinical isolates. Key to the success of this predictive process was the use of liquid chromatography-tandem mass spectrometry to measure the abundance of proteins in extracts of cultured bacteria, identifying which sequence variants seen in the whole-genome sequence data were functionally important in the context of fluoroquinolone susceptibility.

ABSTRACTWe report the characterization of an intrinsic, chromosomally carried aph(3′)-IIcgene from Stenotrophomonas maltophiliaclinical isolate K279a, encoding an aminoglycoside phosphotransferase enzyme that significantly increases MICs of kanamycin, neomycin, butirosin, and paromomycin when expressed in Escherichia coli. Disruption of aph(3′)-IIcin K279a results in decreased MICs of these drugs.

We report the characterization of an intrinsic, chromosomally carried aph(3')-IIc gene from Stenotrophomonas maltophilia clinical isolate K279a, encoding an aminoglycoside phosphotransferase enzyme that significantly increases MICs of kanamycin, neomycin, butirosin, and paromomycin when expressed in Escherichia coli. Disruption of aph(3')-IIc in K279a results in decreased MICs of these drugs.

&lt;it>Objectives&lt;/it>: To determine the level of variation in the &lt;it>smeDEF&lt;/it> efflux pump and &lt;it>smeT&lt;/it> transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical &lt;it>Stenotrophomonas maltophilia&lt;/it> isolates. &lt;it>Methods&lt;/it>: &lt;it>smeDEF&lt;/it> sequencing used a PCR genome walking approach. Determination of the sequence surrounding &lt;it>smeDEF&lt;/it> used a flanking primer PCR method and specific primers anchored in &lt;it>smeD&lt;/it> or &lt;it>smeF&lt;/it> together with random primers. &lt;it>Results&lt;/it>: &lt;it>smeDEF&lt;/it> is chromosomal and located in the same position in the chromosome in all three subgroups of isolates. Flanking &lt;it>smeD&lt;/it> is a gene, &lt;it>smeT&lt;/it>, encoding a putative transcriptional repressor for &lt;it>smeDEF&lt;/it>. Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic β-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint. The &lt;it>smeD&lt;/it>–&lt;it>smeT&lt;/it> intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates. &lt;it>Conclusions&lt;/it>: These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, &lt;it>S. maltophilia&lt;/it>, based on genotypic properties. Isolate D457, in which most work concerning &lt;it>smeDEF&lt;/it> expression has been performed, does not fall into &lt;it>S. maltophilia&lt;/it> subgroup A, which is the most typical.

&lt;it>Objectives&lt;/it>: Isolates previously identified as &lt;it>Citrobacter diversus&lt;/it> are now known as &lt;it>Citrobacter koseri&lt;/it>. We measured sequence variation at the &bgr;-lactamase structural gene among a group of clinical isolates originally identified as &lt;it>C. diversus&lt;/it> by API 20E profiling. &lt;it>Methods&lt;/it>: &bgr;-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods. &bgr;-Lactamase induction was attempted in liquid-grown cultures using cefoxitin. Nitrocefin hydrolysis assays were performed using a spectrophotometer. &lt;it>Results&lt;/it>: Analysis of 16S rRNA gene sequences showed that &lt;it>Citrobacter&lt;/it> spp. isolates with an inducible &bgr;-lactamase gene, &lt;it>cdiA&lt;/it>, closely related to ‘&lt;it>C. koseri&lt;/it> ’ NF85 and ULA27 are actually &lt;it>Citrobacter amalonaticus&lt;/it>. &lt;it>C. koseri&lt;/it> isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A &bgr;-lactamase, Cko, constitutively at low levels. The &lt;it>cko&lt;/it> and &lt;it>cdiA&lt;/it> &bgr;-lactamase genes share &lt;45&percnt; identity. &lt;it>Conclusions&lt;/it>: We have confirmed that &lt;it>cko&lt;/it> is a &bgr;-lactamase gene carried by &lt;it>C. koseri&lt;/it>, and that isolates previously identified as ‘&lt;it>C. koseri&lt;/it> ’, but carrying the c&lt;it>diA&lt;/it> &bgr;-lactamase gene are &lt;it>C. amalonaticus&lt;/it>. Thus, &bgr;-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous &lt;it>Citrobacter&lt;/it> species.

&lt;it>Objectives&lt;/it>: To analyse the variation of &lt;it>ampC&lt;/it> &bgr;-lactamase gene sequence and expression in biochemically atypical Enterobacteriaceae isolates, and to identify them definitively. &lt;it>Methods&lt;/it>: &bgr;-Lactamase gene-containing recombinant plasmids transformed into &lt;it>Escherichia coli&lt;/it> were selected using ampicillin. PCR analysis was used to locate specific &lt;it>ampC&lt;/it> and 16S rRNA genes, and the amplicons were sequenced. Random amplified polymorphic DNA PCR was used to group isolates and API 20E biochemical profiling was used to identify them putatively. &lt;it>Results&lt;/it>: Of 50 ceftazidime-resistant clinical Enterobacteriaceae isolates, 36 were identified (>95&percnt; confidence)—using API 20E test strips—as being organisms known to express inducible class C &bgr;-lactamases (&lt;it>Citrobacter freundii&lt;/it>, &lt;it>Enterobacter cloacae&lt;/it>, &lt;it>Morganella morganii&lt;/it> or &lt;it>Hafnia alvei&lt;/it>). The rest were biochemically atypical. Of these, isolate I113, putatively identified as &lt;it>E. coli&lt;/it>, possesses a chromosomally encoded &lt;it>ampC&lt;/it> which differs by 15&percnt; from &lt;it>C. freundii&lt;/it> OS60 &lt;it>ampC&lt;/it> and by >30&percnt; from &lt;it>E. coli&lt;/it> &lt;it>ampC&lt;/it>. A related &lt;it>ampC&lt;/it> gene was found in another seven of the atypical isolates. The use of various identification methods, including &lt;it>ampC&lt;/it> sequence analysis, revealed that these I113-like &lt;it>ampC&lt;/it>-positive isolates represent &lt;it>Citrobacter murliniae&lt;/it> and &lt;it>Citrobacter youngae&lt;/it>. &lt;it>Conclusions&lt;/it>: We report sequences for two new &lt;it>Citrobacter&lt;/it> spp. &lt;it>ampC&lt;/it> genes, and provide evidence that &lt;it>ampC&lt;/it> sequencing is a discriminatory method for identifying atypical &lt;it>Citrobacter&lt;/it> spp. isolates.

&lt;it>Objectives&lt;/it>: To further understand the mechanisms used to regulate expression of the blr regulon of &lt;it>Aeromonas hydrophila&lt;/it> T429125, including three unlinked &bgr;-lactamase genes, &lt;it>ampH&lt;/it>, &lt;it>cepH&lt;/it> and &lt;it>imiH&lt;/it>, and to examine the role of the ‘cre/blr-tag’ DNA sequence (TTCAC) in transcriptional control exerted by the two-component system, BlrAB. &lt;it>Methods&lt;/it>: Genes linked to &lt;it>blrAB-ampH&lt;/it> were cloned using standard methods; gene expression was measured by RT–PCR or &bgr;-lactamase assays; transcription start sites were determined by reversed-transcript analysis; &lt;it>cepH&lt;/it> promoter probe reporter constructs including cre/blr-tag deletions were generated by PCR; and BlrA was overexpressed in &lt;it>Escherichia coli&lt;/it> using the pBAD plasmid. &lt;it>Results&lt;/it>: The &lt;it>blrD&lt;/it> gene, encoding a putative inner membrane protein, was found to be located downstream of &lt;it>blrAB-ampH&lt;/it>. RT–PCR analysis showed that &lt;it>blrD&lt;/it> is part of the &lt;it>A. hydrophila&lt;/it> blr regulon, and transcript start-point determinations revealed that blr-regulon promoters (including that of &lt;it>blrD&lt;/it>) are preceded by at least one cre/blr-tag. Targeted deletion of the 16 bp &lt;it>cepH&lt;/it> cre/blr-tag dimer blocked BlrA-induced overproduction of &lt;it>cepH&lt;/it> in &lt;it>E. coli&lt;/it>. &lt;it>Conclusions&lt;/it>: This is the first report of non-&bgr;-lactamase genes being co-ordinately regulated with a normally co-resident &bgr;-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.

&lt;it>Aeromonas hydrophila&lt;/it> T429125, a human clinical isolate, possesses three coordinately inducible &bgr;-lactamases encoded by &lt;it>ampH&lt;/it> (class D &bgr;-lactamase), &lt;it>cepH&lt;/it> (class C &bgr;-lactamase) and &lt;it>imiH&lt;/it> (class B &bgr;-lactamase). We report that upstream of &lt;it>ampH&lt;/it> there are two genes, &lt;it>blrA&lt;/it> and &lt;it>blrB&lt;/it>, encoding a putative two-component regulatory system. PCR studies revealed the same &lt;it>blrAB–amp&lt;/it> gene arrangement in all &lt;it>Aeromonas&lt;/it> spp. isolates tested; namely, &lt;it>Aeromonas veronii&lt;/it> bv. sobria, &lt;it>Aeromonas jandaei&lt;/it>, &lt;it>Aeromonas mediae&lt;/it>, &lt;it>Aeromonas salmonicida&lt;/it> and &lt;it>Aeromonas trota&lt;/it>. A dominant mutation in the predicted BlrB kinase domain results in &bgr;-lactamase overexpression in &lt;it>A. hydrophila&lt;/it> T429125, but in other &bgr;-lactamase-overexpressing mutants &lt;it>blrAB&lt;/it> remains intact. Relative to the parent strain, &lt;it>A. hydrophila&lt;/it> T429125, &bgr;-lactamase- overexpressing mutants show a clear hierarchy of increased &bgr;-lactamase expression: ImiH > CepH > AmpH. The same hierarchy is seen following &bgr;-lactam challenge of &lt;it>A. hydrophila&lt;/it> T429125, and correlates with the number of blr-tag sequences (TTCAC) found upstream of each &bgr;-lactamase gene: &lt;it>ampH&lt;/it> (one), &lt;it>cepH&lt;/it> (two) and &lt;it>imiH&lt;/it> (three).