Theodore Gouliouris

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance.

Fecal microbiota transplantation (FMT) for Clostridium difficile infection: Focus on immunocompromised patients

Clostridium difficile infection (CDI) is an emerging problem worldwide associated with significant morbidity, mortality, recurrence rates and healthcare costs. Immunosuppressed patients, including HIV-seropositive individuals, solid organ transplant recipients, patients with malignancies, hematopoietic stem cell transplant recipients, and patients with inflammatory bowel disease are increasingly recognized as being at higher risk of developing CDI where it may be associated with significant complications, recurrence, and mortality. Fecal microbiota transplantation (FMT) has proven to be an effective and safe procedure for the treatment of recurrent or refractory CDI in immunocompetent patients by restoring the gut microbiota and resistance to further recurrences. During the last two years the first data on FMT in immunocompromised patients began to appear in the medical literature. Herein we summarize the use of FMT for the treatment of CDI with a focus on immunocompromised patients.

Complex Routes of Nosocomial Vancomycin-Resistant Enterococcus faecium Transmission Revealed by Genome Sequencing

Summary We generated genome sequence data from 293 Enterococcus faecium isolates from patients with bacteremia spanning 7 years in one hospital. Cases were connected to numerous transmission routes, the complexity of which placed them beyond detection by conventional infection control methods.

Strongyloides stercoralis hyperinfection in a patient treated for multiple myeloma

A 53-year-old Ghanaian man, resident in the UK for many years, was diagnosed with myeloma and commenced treatment with cyclophosphamide, thalidomide and dexamethasone. Towards the end of his first cycle he presented with fever, abdominal pain, constipation and anorexia. There was no recent travel history. Blood tests on admission to hospital showed anaemia (haemoglobin concentration 87 g/l), lymphopenia (white cell count 8·4 9 10/l, lymphocytes 0·7 9 10/l, neutrophils 6·5 9 10/l, eosinophils 0·3 9 10/l) and profound hyponatraemia (plasma sodium 115 mmol/l) as a consequence of the syndrome of inappropriate anti-diuretic hormone release (SIADH). C-reactive protein was mildly raised (24 mg/l). Following an episode of haematemesis, gastroscopy revealed gastritis and duodenitis. Computed tomography (CT) of his abdomen showed diffuse thickening of the entire small bowel and proximal colon (left). He deteriorated clinically with type-one respiratory failure requiring ventilation and intensive care unit (ICU) support. A CT chest scan was consistent with acute respiratory distress syndrome. The diagnosis of Strongyloides stercoralis hyperinfection was made following gastric biopsy, where the filariform larvae of the parasite were seen (right). Parasites were subsequently observed in bronchial washes. Treatment with oral ivermectin was started, but later converted to subcutaneous administration as persistent gastric bleeding abrogated enteric absorption. Slow improvement was made thereafter and the ivermectin dose was gradually tapered. The patient was discharged from hospital on day 42 and remains well. On review of his previous blood results, an unexplained mild eosinophilia (2·2 9 10/l) had been present prior to commencement of chemotherapy. Strongyloides stercoralis infection is endemic in the tropics and subtropics and can persist subclinically for many years. Strongyloides hyperinfection is a serious and well recognized complication of immunosuppression, which is characteristically not accompanied by eosinophilia. Chronic Strongyloides infection should be excluded in patients with eosinophilia originating from, or having travelled to, endemic areas prior to initiating immunosuppressive therapy or steroids. Parenteral ivermectin is unlicensed for human use but is an effective alternative if the oral route is unavailable.

Duration of exposure to multiple antibiotics is associated with increased risk of VRE bacteraemia: a nested case-control study

Abstract Background VRE bacteraemia has a high mortality and continues to defy control. Antibiotic risk factors for VRE bacteraemia have not been adequately defined. We aimed to determine the risk factors for VRE bacteraemia focusing on duration of antibiotic exposure. Methods A retrospective matched nested case-control study was conducted amongst hospitalized patients at Cambridge University Hospitals NHS Foundation Trust (CUH) from 1 January 2006 to 31 December 2012. Cases who developed a first episode of VRE bacteraemia were matched 1:1 to controls by length of stay, year, specialty and ward type. Independent risk factors for VRE bacteraemia were evaluated using conditional logistic regression. Results Two hundred and thirty-five cases were compared with 220 controls. Duration of exposure to parenteral vancomycin, fluoroquinolones and meropenem was independently associated with VRE bacteraemia. Compared with patients with no exposure to vancomycin, those who received courses of 1–3 days, 4–7 days or >7 days had a stepwise increase in risk of VRE bacteraemia [conditional OR (cOR) 1.2 (95% CI 0.4–3.8), 3.8 (95% CI 1.2–11.7) and 6.6 (95% CI 1.9–22.8), respectively]. Other risk factors were: presence of a central venous catheter (CVC) [cOR 8.7 (95% CI 2.6–29.5)]; neutropenia [cOR 15.5 (95% CI 4.2–57.0)]; hypoalbuminaemia [cOR 8.5 (95% CI 2.4–29.5)]; malignancy [cOR 4.4 (95% CI 1.6–12.0)]; gastrointestinal disease [cOR 12.4 (95% CI 4.2–36.8)]; and hepatobiliary disease [cOR 7.9 (95% CI 2.1–29.9)]. Conclusions Longer exposure to vancomycin, fluoroquinolones or meropenem was associated with VRE bacteraemia. Antimicrobial stewardship interventions targeting high-risk antibiotics are required to complement infection control procedures against VRE bacteraemia.

Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing

Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (blaOXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.

Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents

ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars.

The rise and fall of mandatory surveillance for glycopeptide-resistant enterococcal bacteraemia in England

For the treatment of brucellosis, the World Health Organisation (WHO) recommends only a limited number of antibiotics with good intracellular penetration and proven clinical efficiency. Generally, Brucella species are considered susceptible to the WHO recommended antibiotics through sporadic cases of antimicrobial resistance occur. Sensitivity testing is infrequently performed due to concerns over laboratory-acquired infection and the need for containment level 3 facilities. However, in-vitro susceptibilities can vary across geographical regions. Furthermore, until recently invitro susceptibility testing of Brucella spp has not been standardised. Currently, the UK’s British Society of Antimicrobial Chemotherapy and the European Committee on Antimicrobial Susceptibility Testing have not published interpretative breakpoints for brucella antimicrobial sensitivities. As a consequence there are no antimicrobial susceptibility data for Brucella spp cultured from UK patients. We therefore report a prospective study of in-vitro sensitivities to commonly used anti-brucella antibiotic agents. Since 2011, Brucella spp referred to the Brucella Biohazard Facility, Animal Health and Veterinary Agency, for confirmatory identification have routinely undergone antimicrobial sensitivity testing to tetracycline (TET), rifampicin (RIF), ciprofloxacin (CIP), streptomycin (STREP) and gentamicin (GENT) by E-test methodology (Biomerieux, Sweden). The minimum inhibitory concentration (MIC) was interpreted as the value at which the inhibition zone intercepted the scale on the E-strip. MIC90 was defined as the lowest concentration of an antibiotic at which 90% of isolates were inhibited. Clinical Laboratory Standards Institute sensitivity breakpoints (mg/L) were employed for TET (<1), RIF ( 2) and STREP ( 8). For CIP and GENT, breakpoints were set at 2 based on previously published data. Seventeen Brucella melitensis isolates were examined. MIC90 values (mg/l) for TET, RIF, CIP, STREP and GENT were 0.064, 1.0, 0.25, 1.0 and 0.25 respectively. Tetracycline was found to be the most active agent followed by CIP/GENT and then RIF/STREP. No antimicrobial resistant strains were identified. Brucellosis is an uncommon infection in the UK and is typically acquired from countries where there is an endemic problem. This is the first UK study that has examined the antimicrobial sensitivity patterns of B. melitensis isolates cultured from UK patients. The WHO considers tetracycline and its derivatives to be one of the most effective antimicrobials against brucellosis. This study confirms that view and should provide reassurance to UK clinicians who manage this infection. Though no antimicrobial resistance in UK strains was identified, continued surveillance for the emergence of antimicrobial resistance in brucellosis is justified and is ongoing. References

Genome-Based Analysis of Enterococcus faecium Bacteremia Associated with Recurrent and Mixed-Strain Infection

ABSTRACT Vancomycin-resistant Enterococcus faecium (VREfm) bloodstream infections are associated with high recurrence rates. This study used genome sequencing to accurately distinguish the frequency of relapse and reinfection in patients with recurrent E. faecium bacteremia and to investigate strain relatedness in patients with apparent VREfm and vancomycin-susceptible E. faecium (VSEfm) mixed infection. A retrospective study was performed at the Cambridge University Hospitals NHS Foundation Trust (CUH) between November 2006 and December 2012. We analyzed the genomes of 44 E. faecium isolates from 21 patients (26 VREfm isolates from 12 patients with recurrent bacteremia and 18 isolates from 9 patients with putative VREfm/VSEfm mixed infection). Phenotypic antibiotic susceptibility was determined using a Vitek2 instrument. Genomes were compared with those of a further 263 E. faecium isolates associated with bacteremia in patients at CUH over the same time period. Pairwise comparison of core genomes indicated that 10 (71%) episodes of recurrent VREfm bacteremia were due to reinfection with a new strain, with reinfection being more likely with increasing time between the two positive cultures. The majority (78%) of patients with a mixed VREfm and VSEfm infection had unrelated strains. More than half (59%) of study isolates were closely related to another isolate associated with bacteremia from CUH. This included 60% of isolates associated with reinfection, indicating acquisition in the hospital. This study provides the first high-resolution insights into recurrence and mixed infection by E. faecium and demonstrates that reinfection with a new strain, often acquired from the hospital, is a driver of recurrence.

One Health genomic surveillance of Escherichia coli demonstrates distinct lineages and mobile genetic elements in isolates from humans versus livestock

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli (including 155 ESBL-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1517 E. coli associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, indicating that E. coli causing serious human infection do not directly originate from livestock. By contrast, we observed highly related isolates from the same animal species on different farms. Analysis of accessory (variable) genomes identified a virulence cassette associated previously with cystitis and neonatal meningitis that was only present in isolates from humans. Screening all 1948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions of humans and livestock isolates. We identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that in this setting, there was limited evidence to support the suggestion that antimicrobial resistant pathogens that cause serious infection in humans originate from livestock. Importance The increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1517 patients with bloodstream infection were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was little overlap in the mobile elements carrying these genes. In addition, a virulence cassette found in humans isolates was not identified in any livestock-associated isolate. Our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock.