Trends in ExPEC serogroups in the UK and their significance.

Extra-intestinal pathogenic Escherichia coli are a significant cause of urinary tract infection and bacteraemia within the UK. We sought to identify the serogroups of 658 E. coli isolates collected in the UK between January 2011 and March 2012, to better understand the ExPEC population and understand the relevance of serogroups in this pathotype. Isolates were typed and serogroup identified using established phenotypic and molecular methods. Sixty-two serogroups were identified; 54 among urinary isolates and 35 among bloodstream isolates. However, serogroups O25, O6, and O2 dominated both infection types. These serogroups were linked to the major ExPEC STs as follows: ST131-O25, ST73-O6, ST127-O6, and ST95-O2. The serogroup data from this study have increased our understanding of the ExPEC population in the UK, but also highlighted key ST-serogroup relationships within the major ExPEC clones. These data can be used to guide vaccine design and in the development of laboratory diagnostic tests targeting the ExPEC population.

Co-treatment of domestic and dairy wastewater in an activated sludge system.

This research assesses the potential for co-treatment of a dairy wastewater with a domestic wastewater in a lab-scale, continuous-flow, activated sludge system. To evaluate the influence of the dairy waste contribution, seven runs were conducted with each run being a mixture of dairy wastewater (either cheese or milk) in different ratios ranging from 1:0.01 to 1:0.30 by volume. More than 87% of the carbon was removed for both waste additions; however, while 95% ammonia-nitrogen removal was recorded for the cheese waste, only 75% removal was obtained for the milk. Kinetic studies for carbon consumption revealed a first-order model with lower kinetic constants as the cheese waste proportion increased. Specific carbon consumption rates indicated that the biomass had not reached its maximum potential to degrade carbon. The ability of the biomass to settle was hindered when the Gram negative to Gram positive filamentous bacteria ratio increased to approximately 1.5.

Activity of colistin in combination with tigecycline or rifampicin against multidrug-resistant Stenotrophomonas maltophilia.

The antimicrobial treatment of Stenotrophomonas maltophilia infections is complicated by intrinsic multidrug resistance and a lack of reliable susceptibility data. We assessed the activity of colistin (COL), rifampicin (RIF) and tigecycline (TGC) alone and in combination using a range of in vitro susceptibility testing methodologies and a simple invertebrate model of S. maltophilia infection (Galleria mellonella). Synergy [fractional inhibitory concentration indices (FICIs) ≤0.5] between COL and either RIF or TGC was observed against 92 % and 88 % of 25 S. maltophilia isolates, respectively, despite resistance to one or another of the single agents alone. In time-kill assays, COL combined with either RIF or TGC was superior to single agents, but only the COL/RIF regimen was reliably bactericidal. The in vitro findings correlated with treatment outcomes in G. mellonella, with heightened survival observed for larvae treated with COL/RIF or COL/TGC compared with COL, RIF or TGC alone. COL combined with RIF was the most effective combination overall in both in vitro and in vivo (p < 0.05) assays. Given the difficulty in selecting appropriate therapy for S. maltophilia infections, regimens consisting of COL combined with RIF or TGC could be considered for clinical use.

In vitro activity of daptomycin in combination with low-dose colistin against a diverse collection of Gram-negative bacterial pathogens.

The activity of colistin in combination with daptomycin was assessed using 30 Gram-negative type strains and multidrug-resistant isolates with defined mechanisms of resistance. Daptomycin minimum inhibitory concentrations (MICs) were determined with and without sub-inhibitory concentrations of colistin. The activity of daptomycin was not affected with respect to Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa. For colistin-susceptible Acinetobacter baumannii, sensitisation factors ranged from 8 to 128 (median 32), with the daptomycin MIC being reduced to the Clinical and Laboratory Standards Institute (CLSI) enterococci susceptibility breakpoint of 4 µg/ml for the ATCC 19606 type strain. A combination of daptomycin and colistin may be useful for the treatment of A. baumannii but not infections due to other Gram-negative species.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: rapid identification of bacteria isolated from patients with cystic fibrosis.

Despite extensive research into the diagnosis and management of cystic fibrosis (CF) over the past decades, sufferers still have a median life expectancy of less than 37 years. Respiratory tract infections have a significant role in increasing the morbidity and mortality of patients with CF via a progressive decline in lung function. Rapid identification of organisms recovered from CF sputum is necessary for effective management of respiratory tract infections; however, standard techniques of identification are slow, technically demanding and expensive. The aim of this study is to asses the suitability of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) in identifying bacteria isolated from the respiratory tract of patients with CF, and is assessed by testing the accuracy of MALDI-TOF MS in identifying samples from a reference collection of rare CF strains in conjunction with comparing MALDI-TOF MS and standard techniques in identifying clinical isolates from sputum samples of CF patients. MALDI-TOF MS accurately identified 100% of isolates from the reference collection of rare CF pathogens (EuroCare CF collection). The isolate identification given by MALDI-TOF MS agreed with that given by standard techniques for 479/481 (99.6%) clinical isolates obtained from respiratory samples provided by patients with CE In two (0.4%) of 481 samples there was a discrepancy in identification between MALDI-TOF MS and standard techniques. One organism was identified as Pseudomonas aeruginosa by MALDI-TOF but could only be identified by the laboratory's standard methods as of the Pseudomonas genus. The second organism was identified as P. beteli by MALDI-TOF MS and Stenotrophomonas maltophilia by standard methods. This study shows that MALDI-TOF MS is superior to standard techniques in providing cheap, rapid and accurate identification of CF sputum isolates.

In vivo efficacy of glycopeptide-colistin combination therapies in a Galleria mellonella model of Acinetobacter baumannii infection.

The treatment of Acinetobacter baumannii infections poses a significant clinical challenge, with isolates resistant to all commonly used agents increasingly being reported. With few new agents in the pipeline, clinicians are increasingly turning to combinations of antimicrobials in the hope that they may act synergistically together. In this study we assessed the activities of two glycopeptide-colistin combinations both in vitro and using a Galleria mellonella caterpillar model of A. baumannii infection. In checkerboard assays both vancomycin and teicoplanin were highly active against susceptible and multidrug-resistant strains of A. baumannii when combined with colistin (fractional inhibitory concentration [FIC] of <0.25). Treatment of G. mellonella caterpillars infected with lethal doses of A. baumannii resulted in significantly enhanced survival rates when either vancomycin or teicoplanin was given with colistin compared to colistin treatment alone (P < 0.05). This effect was most marked when vancomycin was the glycopeptide administered, although this agent was also highly effective as monotherapy, possibly through an immunomodulatory action on the G. mellonella response to A. baumannii infection. This work suggests that glycopeptide-colistin combinations are highly active against A. baumannii both in vitro and in a simple animal model of infection. They should be considered further as potential treatments for difficult-to-treat A. baumannii infections.

In vitro activity of teicoplanin combined with colistin versus multidrug-resistant strains of Acinetobacter baumannii.

OBJECTIVES: Antimicrobial treatment of multidrug-resistant Acinetobacter baumannii (MDRAB) remains an important therapeutic challenge. With isolates resistant to all conventional agents now reported, clinicians are increasingly forced to turn to unorthodox combination treatments in the hope that these may be efficacious. Although a potent interaction between vancomycin and colistin has been demonstrated, there are concerns regarding the inherent toxicity of combining these agents in clinical practice. As teicoplanin has less nephrotoxic potential than vancomycin, we assessed whether a colistin/teicoplanin combination would have similar antimicrobial activities in vitro. METHODS: The antimicrobial activity of colistin alone and in combination with teicoplanin was assessed versus a collection of MDRAB belonging to a number of epidemic lineages present in the UK. Synergy studies were undertaken using microtitre plate chequerboard assays, an Etest agar dilution method and standard time-kill methodology. RESULTS: The combination of teicoplanin and colistin was bactericidal versus all of the strains tested. In chequerboard assays, fractional inhibitory concentration indices of <0.5 were obtained, consistent with significant in vitro synergy. Using the Etest method the MIC of teicoplanin fell from >256 mg/L to ≤2 mg/L in the presence of subinhibitory concentrations of colistin. CONCLUSIONS: Significant synergy was observed when colistin was combined with teicoplanin versus MDRAB in vitro. This may represent a useful therapeutic combination for the treatment of A. baumannii infections, especially when renal toxicity is a significant concern.

2,4-D removal via denitrification using volatile fatty acids.

Many countries have waters contaminated with both herbicides and nitrates; however, information is limited with respect to removal rates for combined nitrate and herbicide elimination. This research investigates the removal of 2,4-D via denitrification, with a particular emphasis on the effect of adding naturally generated volatile fatty acids (VFAs). The acids were produced from an acid-phase anaerobic digester with a mean VFA concentration of 3153±801 mg/L (as acetic acid). Initially, 2,4-D degrading bacteria were developed in an SBR fed with both sewage and 2,4-D (30-100 mg/L). Subsequent denitrification batch tests demonstrated that the specific denitrification rate increased from 0.0119±0.0039 using 2,4-D alone to 0.0192±0.0079 g NO3-N/g VSS per day, when 2,4-D was combined with natural VFAs from the digester. Similarly, the specific 2,4-D consumption rate increased from 0.0016±0.0009 using 2,4-D alone to 0.0055±0.0021 g 2,4-D/g VSS per day, when using 2,4-D plus natural VFAs. Finally, a parallel increase in the percent 2,4-D removal was observed, rising from 28.33±11.88 using 2,4-D alone to 54.17±21.89 using 2,4-D plus natural VFAs.

Treatment of a slaughterhouse wastewater: effect of internal recycle rate on chemical oxygen demand, total Kjeldahl nitrogen and total phosphorus removal.

This study investigated the ability of an anaerobic/anoxic/oxic (A2/O) system to treat a slaughterhouse wastewater. The system employed two identical continuous-flow reactors (101 total liquid volume each) running in parallel with the main operational variable, being the internal recycle (IR) rate. The chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN) and total phosphorus (TP) performance was evaluated as the IR flowrate was increased from a Q of 151d(-1) to 4Q at a system hydraulic retention time of 16 h and a solids retention time of 10 d. The COD:TKN and COD:TP ratios were 8.2:1 and 54:1, which supported both nitrogen and phosphorus removal. For all IR multiples of Q, the COD removal was in excess of 90%. The TKN removal showed a modest improvement (a 4-5% increase, depending on the dissolved oxygen (DO)) as the IR doubled from Q to 2Q, but no further increase was observed at the 4Q IR rate. The TP removal reached its optimum (around 85%-89% (again depending on the DO)) at the 2Q rate.

Potent synergy and sustained bactericidal activity of a vancomycin-colistin combination versus multidrug-resistant strains of Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (MDRAB) presents an increasing challenge to health care. Although colistin has been used as a treatment of last resort, there is concern regarding its potential for toxicity and the emergence of resistance. The mechanism of action of colistin, however, raises the possibility of synergy with compounds that are normally inactive against Gram-negative organisms by virtue of the impermeability of the bacterial outer membrane. This study evaluated the effect of colistin combined with vancomycin on 5 previously characterized epidemic strains and 34 MDRAB clinical isolates by using time-kill assay, microdilution, and Etest methods. For all the isolates, significant synergy was demonstrated by at least one method, with reductions in the MIC of vancomycin from >256 µg/ml to ≤48 µg/ml for all strains after exposure to 0.5 µg/ml colistin. This raises the possibility of the clinical use of this combination for infections due to MDRAB, with the potential for doses lower than those currently used.

Novel variants of the Smqnr family of quinolone resistance genes in clinical isolates of Stenotrophomonas maltophilia.

BACKGROUND: Recent analysis of Stenotrophomonas maltophilia has identified a novel family of resistance genes (Smqnr) encoding pentapeptide repeat proteins, which confer low-level resistance to quinolones. This study describes further novel variants present in clinical isolates of S. maltophilia and investigates their effect on resistance to a number of quinolones in an Escherichia coli host. METHODS: PCR for Smqnr alleles was carried out on a selection of S. maltophilia from clinical specimens, and amplicons were cloned and transformed in E. coli TOP10 cells. Transformed colonies carrying the plasmid were tested for susceptibility to a range of quinolones by MIC determination. DNA sequences were determined and translated peptide sequences compared with known SmQnr sequences. RESULTS: Thirteen isolates were found to contain Smqnr alleles, of which six corresponded to previously identified Smqnr sequences, while seven were novel variants. Increases in quinolone MICs compared with wild-type E. coli TOP10 were seen for all strains transformed with Smqnr alleles. CONCLUSIONS: There is considerable diversity within Smqnr alleles. S. maltophilia may be a significant reservoir for the dissemination of quinolone resistance elements to Enterobacteriaceae.

Evaluation of CHROMagar Acinetobacter for detection of enteric carriage of multidrug-resistant Acinetobacter baumannii in samples from critically ill patients.

CHROMagar Acinetobacter was used to screen stool and perineal swabs for enteric carriage of multidrug-resistant Acinetobacter baumannii in samples from critically ill patients. Results were compared with a molecular assay resulting in sensitivity and specificity of culture compared to PCR of 91.7% and 89.6%, respectively.

Failure of the MicroScan WalkAway system to detect heteroresistance to carbapenems in a patient with Enterobacter aerogenes bacteremia.

We report the failure of the automated MicroScan WalkAway system to detect carbapenem heteroresistance in Enterobacter aerogenes. Carbapenem resistance has become an increasing concern in recent years, and robust surveillance is required to prevent dissemination of resistant strains. Reliance on automated systems may delay the detection of emerging resistance.

Paradoxical effect of 1-(1-naphthylmethyl)-piperazine on resistance to tetracyclines in multidrug-resistant Acinetobacter baumannii.

OBJECTIVES: The efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP) has been demonstrated to reverse multidrug resistance in Acinetobacter baumannii. We investigated the interaction of NMP with tigecycline and three other tetracyclines on clinical isolates of A. baumannii. METHODS: One hundred and four clinical isolates of Acinetobacter were tested for susceptibility to tigecycline, minocycline, doxycycline and tetracycline by disc diffusion, and tigecycline MICs were determined by Etest, both in the presence and absence of NMP. Tigecycline MICs and zones of inhibition were interpreted using the BSAC guidelines. An OXA carbapenemase multiplex PCR was also performed on each isolate. RESULTS: Mean zones of inhibition for tetracycline, doxycycline and minocycline increased by 11.3%, 22.9% and 11.2%, respectively, in the presence of NMP. In contrast, tigecycline susceptibility was decreased in the presence of NMP, with mean zones of inhibition decreasing by 8.4%. Based on PCR results, all but six isolates belonged to the OXA-23 clone 1. CONCLUSIONS: Susceptibility to tigecycline of the A. baumannii OXA-23 clone 1 prevalent in the UK is reduced (approximately 2-fold) by the presence of the efflux inhibitor NMP. NMP does not have the same effect on susceptibility to other tetracyclines.

A review of clinical and microbiological outcomes following treatment of infections involving multidrug-resistant Acinetobacter baumannii with tigecycline.

OBJECTIVES: Multidrug-resistant Acinetobacter baumannii (MRAB) is an increasing problem in UK hospitals, with many strains now resistant to all available antibiotics except polymyxins. Tigecycline has been used for the treatment of MRAB as it demonstrates activity in vitro, but there are limited data on its clinical efficacy in Gram-negative infections, especially those involving the lower respiratory tract or bacteraemia. PATIENTS AND METHODS: A retrospective study of the clinical and microbiological outcomes of all patients treated with tigecycline for MRAB over an 18 month period was undertaken. RESULTS: Thirty-four patients received tigecycline for MRAB or polymicrobial infection involving MRAB. Twenty-three (68%) had a positive clinical outcome: microbiological clearance was demonstrated in 10 of these. The overall mortality was 41% (n = 14), with nine deaths directly attributable to sepsis. Three patients had episodes of Gram-negative bacteraemia while receiving treatment with tigecycline, with documented resistance occurring in one patient. Overall, the correlation between microbiological and clinical outcomes was poor. CONCLUSIONS: While tigecycline retains excellent in vitro activity against MRAB, its clinical efficacy remains uncertain. A prospective study, including the use of tigecycline in combination with other antimicrobial agents, should be undertaken to define its role in the treatment of MRAB.

Treatment of four industrial wastewaters by sequencing batch reactors: evaluation of COD, TKN and TP removal.

This study investigated the ability of a sequencing batch reactor (SBR) system to treat four industrial wastewaters, namely, textile, landfill leachate, seafood and slaughterhouse effluents. The system employed three identical SBRs (10 l volume each) operating in parallel and each waste was treated one at a time. The operational variables examined included the length of the non-aerated period and the solids retention time (SRT). All four wastewaters experienced chemical oxyfen demand (COD) and total kjeldhal nitrogen (TKN) removals greater than 81%, while the TP removals were lower, ranging from 57 to 94%. The length of the non-aerated period appeared to have minimal effect on the SBR performance; however, increases in SRT reduced the percent TP removal for the textile and leachate wastes only. In addition, to investigate organic loading limits to the seafood SBR system, the COD was increased by three increments of 250 mg l(-1) starting from a baseline concentration of 1100 mg l(-1). This resulted in a reduction in both the TKN and TP removal at the higher concentrations. Finally, for the slaughterhouse wastewater, the COD:TKN ratio was tested at levels of 6:1, 8:1 and 9:1 with the result that only the TP removal was affected at the lowest ratio.

Comparative activity of tedizolid and glycopeptide combination therapies for the treatment of Staphylococcus aureus infections: an in vitro and in vivo evaluation against strains with reduced susceptibility to glycopeptides.

PURPOSE: Glycopeptides are widely used for the treatment of meticillin-resistant Staphylococcus aureus (MRSA) infections. Although difficult to detect, isolates with reduced (GISA), hetero (hGISA) or complete (GRSA) resistance to glycopeptides are increasingly reported. Optimal therapy for such strains is unknown. We compared the in vitro and in vivo activity of tedizolid (TED), a recently licensed oxazolidonone, with vancomycin (VAN) and teicoplanin (TEIC) combined with fusidic acid (FD) or rifampicin (RIF) against S. aureus (SA) with reduced susceptibility to glycopeptides. METHODS: Susceptibility was determined for six (GISA, hGISA and GRSA) reference strains and 72 clinical MRSA isolates screened for hGISA/GISA-like phenotypes. Synergy and bactericidal activity were assessed using chequerboard and time-kill assays. The G. mellonella wax moth caterpillar model was used to measure the activity of TED and the combinations in vivo. RESULTS: Glycopeptide MICs (VAN/TEIC) ranged from 0.5-8/4 and 0.125-1 for TED. No significant synergy was noted when VAN/TEIC were combined with either RIF or FD. Time-kill assays confirmed that TED was bacteriostatic but superior to VAN and TEIC against GISA strains. In G. mellonella TED was more effective than TEIC monotherapy versus GISA strains. The combination of TEIC with RIF was the most effective combination overall, both in vitro and in vivo. CONCLUSIONS: TED had good in vitro activity versus MRSA including those with reduced susceptibility to glycopeptides. Although bacteriostatic, it was effective in the G. mellonella model and superior to TEIC in the treatment of GISA. Although this supports the use of TED for MRSA and GISA, the TEIC/RIF combination also warrants further study.

Optimal detection of carbapenemase-producing Enterobacteriaceae from rectal samples: a role for enrichment?

BACKGROUND: Successful laboratory detection of carbapenemase-producing Enterobacteriaceae (CPE) in patient surveillance samples is a diagnostic challenge. In the absence of a reference standard for screening rectal swabs for CPE, many phenotypic, genotypic, culture- and non-culture-based assays have been proposed for identifying these bacteria. AIM: To develop and optimize a CPE screening protocol capable of identifying all frequently encountered CPE, including those producing OXA-48-like carbapenemases. METHODS: Faropenem susceptibility testing was performed on 507 presumptive CPE isolated from diagnostic samples and CPE rectal screens between March and August 2016. Results from this CPE screening method were compared to those from direct culture on mSuperCARBA™, temocillin enrichment culture, and use of an antibiotic resistance algorithm, to determine the optimal method to employ in the detection of CPE. FINDINGS: Faropenem was a poor predictor of carbapenemase production (58% true positives). The combination of a temocillin enrichment stage and interpretive reading of antibiotic resistance phenotypes improved the recovery and identification of CPE significantly (91% true positives), especially for OXA-48 producers (P = 0.03). CONCLUSION: The combination of temocillin enrichment, a selective chromogenic medium, and an antibiotic resistance-based algorithm significantly improved the detection of all CPE recovered from routine and targeted surveillance samples.

Genetic analysis of mechanisms of multidrug resistance in a clinical isolate of Bacteroides fragilis.

This study investigated the mechanisms of multidrug resistance (MDR) in an isolate of Bacteroides fragilis (WI1) from a patient with anaerobic sepsis. The MDR of WI1 affected susceptibility to beta-lactams, clindamycin, fluoroquinolones, metronidazole and tetracycline. In addition to its 5.31-Mb chromosome, WI1 possessed two low-copy-number plasmids, pHagl (5.6 kb) and pHag2 (9.9 kb), that were absent from B. fragilis NCTC 9343. Restriction digestion with EcoRV, HindIII and SstI, combined with DNA sequencing, revealed that pHAG2 contained a tet(Q) gene at base position 3689 that resided on the conjugative transposon CTn341. Genes cfiA (encoding a metallo-beta-lactamase) and erm(F) (encoding a macrolide-lincosamide-streptogramin B resistance determinant) were also found in WI1, but were absent from B. fragilis NCTC 9343. Nitrocefin hydrolysis revealed that WI1 had high beta-lactamase activity. Sequencing of the gyrA quinolone resistance-determining region revealed a mutation causing a Ser82 --> Phe substitution, and comparative quantitative real-time RT-PCR revealed that the cfiA, erm(F) and tet(Q) genes were all expressed in WI1. In addition, the resistance-nodulation-division efflux pump genes bmeB9 and bmeB15 were significantly over-expressed (12.30 +/- 0.42-fold and 3541.1 +/- 95.4-fold, respectively), and the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone and reserpine significantly increased the susceptibility of the isolate to several unrelated antibiotics (p <0.005). These data suggested that WI1 was highly multidrug-resistant because of the additive effects of chromosome- and plasmid-encoded resistance determinants.

Draft Genome Sequence of Providencia stuartii PS71, a Multidrug-Resistant Strain Associated with Nosocomial Infections in Greece.

Providencia stuartii is frequently associated with nosocomial outbreaks and displays intrinsic resistance to many commonly used antimicrobials. We report here the draft genome sequence of a P. stuartii strain carrying acquired resistance genes conferring panresistance to cephalosporins (blaSHV-5 and blaVEB-1), carbapenems (blaVIM-1), and aminoglycosides (rmtB) involved in an outbreak in Greek hospitals.