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1.  Invasive Adenocarcinoma of the Lung is Associated with the Upper Lung Regions 
We postulated that ventilation-perfusion (V/Q) relationships within the lung might influence where lung cancer occurs. To address this hypothesis we evaluated the location of lung adenocarcinoma, by both tumor lobe and superior-inferior regional distribution, and associated variables such as emphysema.
Materials and Methods
One hundred fifty-nine cases of invasive adenocarcinoma and adenocarcinoma with lepidic features were visually evaluated to identify lobar or regional tumor location. Regions were determined by automated division of the lungs into three equal volumes: (upper region, middle region, or lower region). Automated densitometry was used to measure radiographic emphysema.
The majority of invasive adenocarcinomas occurred in the upper lobes (69%), with 94% of upper lobe adenocarcinomas occurring in the upper region of the lung. The distribution of adenocarcinoma, when classified as upper or lower lobe, was not different between invasive adenocarcinoma and adenocarcinoma with lepidic features (formerly bronchioloalveolar cell carcinoma, P=0.08). Regional distribution of tumor was significantly different between invasive adenocarcinoma and adenocarcinoma with lepidic features (P = 0.001). Logistic regression analysis with the outcome of invasive adenocarcinoma histology was used to adjust for confounders. Tumor region continued to be a significant predictor (OR 8.5, P=0.008, compared to lower region), whereas lobar location of tumor was not (P=0.09). In stratified analysis, smoking was not associated with region of invasive adenocarcinoma occurrence (p=0.089). There was no difference in total emphysema scores between invasive adenocarcinoma cases occurring in each of the three regions (P=0.155). There was also no difference in the distribution of region of adenocarcinoma occurrence between quartiles of emphysema (P=0.217).
Invasive adenocarcinoma of the lung is highly associated with the upper lung regions. This association is not related to smoking, history of COPD, or total emphysema. The regional distribution of invasive adenocarcinoma may be due to V/Q relationships or other local factors.
PMCID: PMC4004700  PMID: 24598367
Non-small cell lung cancer; tumor location; emphysema
2.  Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing 
PeerJ  2015;3:e739.
The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.
PMCID: PMC4338798  PMID: 25737808
Stx; Genomics; Sequencing; O157; E. coli
3.  Enteroaggregative Escherichia coli Have Evolved Independently as Distinct Complexes within the E. coli Population with Varying Ability to Cause Disease 
PLoS ONE  2014;9(11):e112967.
Enteroaggregative E. coli (EAEC) is an established diarrhoeagenic pathotype. The association with virulence gene content and ability to cause disease has been studied but little is known about the population structure of EAEC and how this pathotype evolved. Analysis by Multi Locus Sequence Typing of 564 EAEC isolates from cases and controls in Bangladesh, Nigeria and the UK spanning the past 29 years, revealed multiple successful lineages of EAEC. The population structure of EAEC indicates some clusters are statistically associated with disease or carriage, further highlighting the heterogeneous nature of this group of organisms. Different clusters have evolved independently as a result of both mutational and recombination events; the EAEC phenotype is distributed throughout the population of E. coli.
PMCID: PMC4240581  PMID: 25415318
4.  Evidence of Evolving Extraintestinal Enteroaggregative Escherichia coli ST38 Clone 
Emerging Infectious Diseases  2014;20(11):1935-1937.
PMCID: PMC4214294  PMID: 25340736
Escherichia coli; enteroaggregative; ESBL; extended-spectrum β-lactamase; extraintestinal; ST38; diarrheagenic E. coli; multiple drug resistance; Germany; the Netherlands; United Kingdom; bacteria
5.  Risk factor analysis for the recurrence of resected solitary fibrous tumours of the pleura: a 33-year experience and proposal for a scoring system† 
Surveillance after resection of solitary fibrous tumours of the pleura (SFTP) remains undefined. This study reviews our experience with surgical treatment of SFTP to determine the specific risk factors to predict recurrence.
A retrospective review of 59 patients surgically treated for SFTP during the years 1977–2010 was conducted. Clinico-pathological factors for recurrence were analysed by Kaplan–Meier and Cox proportional hazard methods.
The mean age was 57 ± 14 years. There were 32 (54%) men. Among 32 (54%) symptomatic patients, chest pain (22%), cough (19%) and dyspnoea (17%) were most frequent. The mean tumour size was 7.3 ± 6.7 cm, and 14 patients had SFTPs larger than 10 cm. An SFTP was pedunculated in 38 (67%) cases and had a visceral origin in 40 (68%). Paraneoplastic syndromes were observed in 3 (5%) patients. On histopathologic analysis, 4 (7%) presented ≥4 mitosis/10 high-power fields (HPFs), 8 (15%) atypia, 14 (24%) hypercellularity and 6 (10%) necrosis. After a mean follow-up of 8.8 ± 7.0 years, we observed 8 (14%) recurrences; median time to recurrence was 6 years (range 2–16 years). Two (3%) patients received adjuvant therapy. We constructed a predictive score for recurrence by assigning one point to each of the six variables: parietal (vs visceral) pleural origin, sessile (vs pedunculated) morphology, size >10 cm (vs <10 cm), the presence of hypercellularity, necrosis and mitotic activity ≥4/HPF (vs <4). A score of ≥3 best predicted recurrence (sensitivity: 100%, specificity: 92%, area under receiver operating characteristic curve = 0.966, P < 0.0001). With a score of ≥3, recurrence-free survival was 80%, 69, 23 and 23% at 3, 5, 10 and 15 years, whereas a score of <3 was 100% up to 15 years. Our scoring system was superior in predicting malignant behaviour and recurrence compared with England's criteria or de Perrot staging.
The proposed scoring system is simple, easily obtained from existing pathological description and reliably predicts recurrence in this patient population harbouring SFTP. The SFTP score may stratify patient risk and guide postoperative surveillance. We recommend validation in additional clinical series.
PMCID: PMC3681536  PMID: 23233072
Solitary fibrous tumour; Pleura; Recurrence
7.  An Investigation of the Diversity of Strains of Enteroaggregative Escherichia coli Isolated from Cases Associated with a Large Multi-Pathogen Foodborne Outbreak in the UK 
PLoS ONE  2014;9(5):e98103.
Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.
PMCID: PMC4028294  PMID: 24844597
8.  Short And Long-Term Outcomes After Esophagectomy For Cancer In Elderly Patients 
The Annals of thoracic surgery  2013;95(5):1741-1748.
As worldwide life expectancy rises, the number of candidates for surgical treatment of esophageal cancer over 70 years will increase. This study aims to examine outcomes after esophagectomy in elderly patients.
Retrospective review of 474 patients undergoing esophagectomy for cancer during 2002–2011. 334 (70.5%) patients were <70 years old (group A), 124 (26.2%) 70–79 years (group B) and 16 (3.4%) ≥80 years (group C). We analyzed the effect of age on outcome variables including overall and disease specific survival.
Major morbidity was observed to occur in 115 (35.6%) patients of group A, 58 (47.9%) of group B and 10 (62.5%) of group C (p=0.010). Mortality, both 30- and 90-day was observed in 2(0.6%) and 7(2.2%) of group A, 4(3.2%) and 7 (6.1%) of group B, and 1(6.3%) and 2(14.3%) of group C, respectively (p=0.032 and p=0.013). Anastomotic leak was observed in 16(4.8%) patients of group A, 6(4.8%) of group B and 0(0%) of group C (p=0.685). Anastomotic stricture (defined by the need for ≥2 dilations) was observed in 76(22.8%) of group A, 13(10.5%) of group B and 1(6.3%) of group C (p=0.005). Five-year overall and disease specific survival was 64.8% and 72.4% for group A, 41.7% and 53.4% for group B, 49.2% and 49.2% for group C patients (p=0.0006), respectively.
Esophagectomy should be carefully considered in patients 70–79 years old and can be justified with low mortality. Outcomes in octogenarians are worse suggesting esophagectomy be considered on a case by case basis. Stricture rate is inversely associated to age.
PMCID: PMC3732120  PMID: 23500043
Esophagus; Esophageal cancer; Esophageal surgery; Outcomes; Statistics-regression analysis
9.  Epidemiological Evidence That Garden Birds Are a Source of Human Salmonellosis in England and Wales 
PLoS ONE  2014;9(2):e88968.
The importance of wild bird populations as a reservoir of zoonotic pathogens is well established. Salmonellosis is a frequently diagnosed infectious cause of mortality of garden birds in England and Wales, predominantly caused by Salmonella enterica subspecies enterica serovar Typhimurium definitive phage types 40, 56(v) and 160. In Britain, these phage types are considered highly host-adapted with a high degree of genetic similarity amongst isolates, and in some instances are clonal. Pulsed field gel electrophoresis, however, demonstrated minimal variation amongst matched DT40 and DT56(v) isolates derived from passerine and human incidents of salmonellosis across England in 2000–2007. Also, during the period 1993–2012, similar temporal and spatial trends of infection with these S. Typhimurium phage types occurred in both the British garden bird and human populations; 1.6% of all S. Typhimurium (0.2% of all Salmonella) isolates from humans in England and Wales over the period 2000–2010. These findings support the hypothesis that garden birds act as the primary reservoir of infection for these zoonotic bacteria. Most passerine salmonellosis outbreaks identified occurred at and around feeding stations, which are likely sites of public exposure to sick or dead garden birds and their faeces. We, therefore, advise the public to practise routine personal hygiene measures when feeding wild birds and especially when handling sick wild birds.
PMCID: PMC3935841  PMID: 24586464
10.  Risk factors for the development of severe typhoid fever in Vietnam 
Typhoid fever is a systemic infection caused by the bacterium Salmonella enterica serovar Typhi. Age, sex, prolonged duration of illness, and infection with an antimicrobial resistant organism have been proposed risk factors for the development of severe disease or fatality in typhoid fever.
We analysed clinical data from 581 patients consecutively admitted with culture confirmed typhoid fever to two hospitals in Vietnam during two periods in 1993–1995 and 1997–1999. These periods spanned a change in the antimicrobial resistance phenotypes of the infecting organisms i.e. fully susceptible to standard antimicrobials, resistance to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole (multidrug resistant, MDR), and intermediate susceptibility to ciprofloxacin (nalidixic acid resistant). Age, sex, duration of illness prior to admission, hospital location and the presence of MDR or intermediate ciprofloxacin susceptibility in the infecting organism were examined by logistic regression analysis to identify factors independently associated with severe typhoid at the time of hospital admission.
The prevalence of severe typhoid was 15.5% (90/581) and included: gastrointestinal bleeding (43; 7.4%); hepatitis (29; 5.0%); encephalopathy (16; 2.8%); myocarditis (12; 2.1%); intestinal perforation (6; 1.0%); haemodynamic shock (5; 0.9%), and death (3; 0.5%). Severe disease was more common with increasing age, in those with a longer duration of illness and in patients infected with an organism exhibiting intermediate susceptibility to ciprofloxacin. Notably an MDR phenotype was not associated with severe disease. Severe disease was independently associated with infection with an organism with an intermediate susceptibility to ciprofloxacin (AOR 1.90; 95% CI 1.18-3.07; p = 0.009) and male sex (AOR 1.61 (1.00-2.57; p = 0.035).
In this group of patients hospitalised with typhoid fever infection with an organism with intermediate susceptibility to ciprofloxacin was independently associated with disease severity. During this period many patients were being treated with fluoroquinolones prior to hospital admission. Ciprofloxacin and ofloxacin should be used with caution in patients infected with S. Typhi that have intermediate susceptibility to ciprofloxacin.
PMCID: PMC3923984  PMID: 24512443
Salmonella enterica serovar Typhi; Severe typhoid; Antimicrobial resistance; Multidrug resistance; Intermediate ciprofloxacin susceptibility
11.  Immunological monitoring to prevent and treat sepsis 
Critical Care  2013;17(1):109.
The clinical, human and economic burden associated with sepsis is huge. Initiatives such as the Surviving Sepsis Campaign aim to effectively reduce risk of death from severe sepsis and septic shock. Nonetheless, although substantial benefits raised from the implementation of this campaign have been obtained, much work remains if we are to realise the full potential promised by this strategy. A deeper understanding of the processes leading to sepsis is necessary before we can design an effective suite of interventions. Dysregulation of the immune response to infection is acknowledged to contribute to the pathogenesis of the disease. Production of both proinflammatory and immunosuppressive cytokines is observed from the very first hours following diagnosis. In addition, hypogammaglobulinemia is often present in patients with septic shock. Moreover, levels of IgG, IgM and IgA at diagnosis correlate directly with survival. In turn, nonsurvivors have lower levels of C4 (a protein of the complement system) than the survivors. Natural killer cell counts and function also seem to have an important role in this disease. HLA-DR in the surface of monocytes and counts of CD4+CD25+ T-regulatory cells in blood could also be useful biomarkers for sepsis. At the genomic level, repression of networks corresponding to major histocompatibility complex antigen presentation is observed in septic shock. In consequence, cumulative evidence supports the potential role of immunological monitoring to guide measures to prevent or treat sepsis in a personalised and timely manner (early antibiotic administration, immunoglobulin replacement, immunomodulation). In conclusion, although diffuse and limited, current available information supports the development of large comprehensive studies aimed to urgently evaluate immunological monitoring as a tool to prevent sepsis, guide its treatment and, as a consequence, diminish the morbidity and mortality associated with this severe condition.
PMCID: PMC4057291  PMID: 23351425
12.  Evaluating the Use of Multilocus Variable Number Tandem Repeat Analysis of Shiga Toxin-Producing Escherichia coli O157 as a Routine Public Health Tool in England 
PLoS ONE  2014;9(1):e85901.
Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.
PMCID: PMC3895024  PMID: 24465775
13.  Complete Genome Sequence of the Campylobacter coli Clinical Isolate 15-537360 
Genome Announcements  2013;1(6):e01056-13.
Campylobacter coli strain 15-537360 was originally isolated in 2001 from a 42-year-old patient with gastroenteritis. Here, we report its complete genome sequence, which comprises a 1.7-Mbp chromosome and a 29-kbp conjugative cryptic plasmid. This is the first complete genome sequence of a clinical isolate of C. coli.
PMCID: PMC3861437  PMID: 24336384
14.  In Vivo Imaging of Tracheal Epithelial Cells in Mice during Airway Regeneration 
Many human lung diseases, such as asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans, and cystic fibrosis, are characterized by changes in the cellular composition and architecture of the airway epithelium. Intravital fluorescence microscopy has emerged as a powerful approach in mechanistic studies of diseases, but it has been difficult to apply this tool for in vivo respiratory cell biology in animals in a minimally invasive manner. Here, we describe a novel miniature side-view confocal probe capable of visualizing the epithelium in the mouse trachea in vivo at a single-cell resolution. We performed serial real-time endotracheal fluorescence microscopy in live transgenic reporter mice to view the three major cell types of the large airways, namely, basal cells, Clara cells, and ciliated cells. As a proof-of-concept demonstration, we monitored the regeneration of Clara cells over 18 days after a sulfur dioxide injury. Our results show that in vivo tracheal microscopy offers a new approach in the study of altered, regenerating, or metaplastic airways in animal models of lung diseases.
PMCID: PMC3547097  PMID: 22984086
in vivo fluorescence microscopy; mouse imaging; epithelial regeneration
15.  Genomic Characterisation of Invasive Non-Typhoidal Salmonella enterica Subspecies enterica Serovar Bovismorbificans Isolates from Malawi 
Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004.
We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains.
Principal Findings
Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK.
The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov).
iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.
Author Summary
Bacteraemia and meningitis caused by non-typhoidal Salmonella (including serovars Typhimurium, Enteritidis and Bovismorbificans) are a serious health issue in sub-Saharan Africa, particularly in young children and HIV-infected adults. Previous work has indicated that a distinct S. Typhimurium sequence type, ST313, has evolved and spread in these countries, and may be more human-adapted than isolates found in the developed world. We therefore investigated the genomes of Salmonella enterica serovar Bovismorbificans bacteraemia isolates from Malawi and compared them to genomes of veterinary S. Bovismorbificans isolates from the UK using Next Generation Sequencing Technology and subsequent genomic comparisons to establish if there is a genetic basis for this increase in invasive disease observed among African NTS. Contrary to the previous findings for S. Typhimurium, where a distinct ST is found only in sub-Saharan Africa, we discovered that the S. Bovismorbificans isolates from Malawi belong to the most common ST of the serovar and the genome is highly conserved across all sequenced isolates. The major differences between UK veterinary and African human isolates were due to prophage regions inserted into the genomes of African isolates, coupled with a higher prevalence of a virulence plasmid compared to the UK isolates.
PMCID: PMC3828162  PMID: 24244782
16.  Mechanical Ventilation and Air Leaks After Lung Biopsy for Acute Respiratory Distress Syndrome 
The Annals of thoracic surgery  2006;82(1):10.1016/j.athoracsur.2006.02.022.
Open lung biopsy in acute respiratory distress syndrome (ARDS) may provide a specific etiology and change clinical management, yet concerns about complications remain. Persistent air leak is the most common postoperative complication. Risk factors in this setting are not known.
We performed a retrospective analysis of 53 patients who underwent open lung biopsy for clinical ARDS (based on American European Consensus Conference criteria) between 1989 and 2000.
Sixteen patients (30.2%) developed an air leak lasting more than 7 days or died with an air leak. Univariate analyses showed no significant correlation with age, gender, sex, corticosteroid use, diabetes, immunocompromised status, or pathologic diagnosis. A lower risk of air leak was associated with lower peak airway pressure and tidal volume, use of pressure-cycled ventilation, and use of an endoscopic stapling device. In multivariate analyses, only peak airway pressure remained a significant predictor. The risk of prolonged air leak was reduced by 42% (95% confidence interval [CI: 17% to 60%]) for every 5 cm H2O reduction in peak airway pressure.
The use of a lung-protective ventilatory strategy that limits peak airway pressures is strongly associated with a reduced risk of postoperative air leak after open lung biopsy in ARDS. Using such a strategy may allow physicians to obtain information from open lung biopsy to make therapeutic decisions without undue harm to ARDS patients.
PMCID: PMC3822769  PMID: 16798226
17.  Comparative Analysis of ESBL-Positive Escherichia coli Isolates from Animals and Humans from the UK, The Netherlands and Germany 
PLoS ONE  2013;8(9):e75392.
The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.
PMCID: PMC3784421  PMID: 24086522
18.  MTHFR Polymorphisms, Folate Intake, and Carcinogen DNA Adducts in the Lung 
The methylenetetrahydrofolate reductase (MTHFR) genes and folate in one-carbon metabolism are essential for DNA methylation and synthesis. However, their role in carcinogen DNA damage in target lung tissue, a dosimeter for cancer risk, is not known. Our study aimed to investigate the association between genetic and nutritional one-carbon metabolism factors and DNA adducts in target lung. Data on 135 lung cancer cases from the Massachusetts General Hospital were studied. Genotyping was completed for MTHFR C677T (rs1801133) and A1298C (rs1801131). Information on dietary intake for one-carbon related micronutrients, folate and other B vitamin, was derived from a validated food frequency questionnaire. DNA adducts in lung were measured by 32P-postlabeling. After adjusting for potential confounders, DNA adduct levels in lung significantly increased by 69.2% [95% confidence interval (CI), 5.5% to 171.5%] for the MTHFR 1298AC+CC genotype. The high risk group, combining the A1298C (AC+CC) plus C677T (CT+TT) genotypes, had significantly enhanced levels of lung adducts by 210.7% (95% CI, 21.4% to 695.2%) in contrast to the A1298C (AA) plus C677T (CC) genotypes. Elevation of DNA adduct was pronounced - 111.3% (95% CI, −3.0 to 360.5%) among 1298AC+CC patients who consumed the lowest level of folate intake as compared with 1298AA individuals with highest tertile of intake. These results indicate that DNA adducts levels are influenced by MTHFR polymorphisms and low folate consumption, suggesting an important role of genetic and nutritional factors in protecting DNA damage from lung carcinogen in at-risk populations.
PMCID: PMC3293105  PMID: 22052259
MTHFR; folate; genetic polymorphisms; DNA adducts; one carbon metabolism
19.  Public Health Value of Next-Generation DNA Sequencing of Enterohemorrhagic Escherichia coli Isolates from an Outbreak 
Journal of Clinical Microbiology  2013;51(1):232-237.
In 2009, an outbreak of enterohemorrhagic Escherichia coli (EHEC) on an open farm infected 93 persons, and approximately 22% of these individuals developed hemolytic-uremic syndrome (HUS). Genome sequencing was used to investigate outbreak-derived animal and human EHEC isolates. Phylogeny based on the whole-genome sequence was used to place outbreak isolates in the context of the overall E. coli species and the O157:H7 sequence type 11 (ST11) subgroup. Four informative single nucleotide polymorphisms (SNPs) were identified and used to design an assay to type 122 other outbreak isolates. The SNP phylogeny demonstrated that the outbreak strain was from a lineage distinct from previously reported O157:H7 ST11 EHEC and was not a member of the hypervirulent clade 8. The strain harbored determinants for two Stx2 verotoxins and other putative virulence factors. When linked to the epidemiological information, the sequence data indicate that gross contamination of a single outbreak strain occurred across the farm prior to the first clinical report of HUS. The most likely explanation for these results is that a single successful strain of EHEC spread from a single introduction through the farm by clonal expansion and that contamination of the environment (including the possible colonization of several animals) led ultimately to human cases.
PMCID: PMC3536255  PMID: 23135946
20.  Population Genetic Structure of 4,12:a:− Salmonella enterica Strains from Harbor Porpoises 
Applied and Environmental Microbiology  2012;78(24):8829-8833.
According to pulsed-field gel electrophoresis (PFGE) typing, 4,12:a:− Salmonella enterica isolates from harbor porpoises are highly diverse. However, porpoise isolates belong to only two multilocus sequence types within the eBurst group 18 (eBG18) genetic cluster, which also includes S. enterica serovars Bispebjerg and Abortusequi. Isolates of other, serologically similar serovars belong to unrelated eBGs. These assignments to eBGs were supported by eBG-specific sequences of the flagellar gene fliC.
PMCID: PMC3502922  PMID: 23042176
21.  Intra-continental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa 
Nature genetics  2012;44(11):1215-1221.
A highly invasive form of non-typhoidal Salmonella (iNTS) disease has been recently documented in many countries in sub-Saharan Africa. The most common Salmonella enterica serovar causing this disease is Typhimurium. We applied whole-genome sequence-based phylogenetic methods to define the population structure of sub-Saharan African invasive Salmonella Typhimurium and compared these to global Salmonella Typhimurium isolates. Notably, the vast majority of sub-Saharan invasive Salmonella Typhimurium fell within two closely-related, highly-clustered phylogenetic lineages that we estimate emerged independently ~52 and ~35 years ago, in close temporal association with the current HIV pandemic. Clonal replacement of isolates of lineage I by lineage II was potentially influenced by the use of chloramphenicol for the treatment of iNTS disease. Our analysis suggests that iNTS disease is in part an epidemic in sub-Saharan Africa caused by highly related Salmonella Typhimurium lineages that may have occupied new niches associated with a compromised human population and antibiotic treatment.
PMCID: PMC3491877  PMID: 23023330
22.  Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs 
Cell stem cell  2012;10(4):385-397.
Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.
PMCID: PMC3474327  PMID: 22482504
23.  A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium 
Nucleic Acids Research  2013;41(8):4549-4564.
Salmonella Typhi and Typhimurium diverged only ∼50 000 years ago, yet have very different host ranges and pathogenicity. Despite the availability of multiple whole-genome sequences, the genetic differences that have driven these changes in phenotype are only beginning to be understood. In this study, we use transposon-directed insertion-site sequencing to probe differences in gene requirements for competitive growth in rich media between these two closely related serovars. We identify a conserved core of 281 genes that are required for growth in both serovars, 228 of which are essential in Escherichia coli. We are able to identify active prophage elements through the requirement for their repressors. We also find distinct differences in requirements for genes involved in cell surface structure biogenesis and iron utilization. Finally, we demonstrate that transposon-directed insertion-site sequencing is not only applicable to the protein-coding content of the cell but also has sufficient resolution to generate hypotheses regarding the functions of non-coding RNAs (ncRNAs) as well. We are able to assign probable functions to a number of cis-regulatory ncRNA elements, as well as to infer likely differences in trans-acting ncRNA regulatory networks.
PMCID: PMC3632133  PMID: 23470992
24.  Revolutionising Bacteriology to Improve Treatment Outcomes and Antibiotic Stewardship 
Infection & Chemotherapy  2013;45(1):1-10.
Laboratory investigation of bacterial infections generally takes two days: one to grow the bacteria and another to identify them and to test their susceptibility. Meanwhile the patient is treated empirically, based on likely pathogens and local resistance rates. Many patients are over-treated to prevent under-treatment of a few, compromising antibiotic stewardship. Molecular diagnostics have potential to improve this situation by accelerating precise diagnoses and the early refinement of antibiotic therapy. They include: (i) the use of 'biomarkers' to swiftly distinguish patients with bacterial infection, and (ii) molecular bacteriology to identify pathogens and their resistance genes in clinical specimens, without culture. Biomarker interest centres on procalcitonin, which has given good results particularly for pneumonias, though broader biomarker arrays may prove superior in the future. PCRs already are widely used to diagnose a few infections (e.g. tuberculosis) whilst multiplexes are becoming available for bacteraemia, pneumonia and gastrointestinal infection. These detect likely pathogens, but are not comprehensive, particularly for resistance genes; there is also the challenge of linking pathogens and resistance genes when multiple organisms are present in a sample. Next-generation sequencing offers more comprehensive profiling, but obstacles include sensitivity when the bacterial load is low, as in bacteraemia, and the imperfect correlation of genotype and phenotype. In short, rapid molecular bacteriology presents great potential to improve patient treatments and antibiotic stewardship but faces many technical challenges; moreover it runs counter to the current nostrum of defining resistance in pharmacodynamic terms, rather than by the presence of a mechanism, and the policy of centralising bacteriology services.
PMCID: PMC3780945  PMID: 24265945
Biomarkers; Molecular microbiology; Rapid diagnostics; Antibiotic stewardship
25.  The TCA cycle is not required for selection or survival of multidrug-resistant Salmonella 
The initial aim of this study was to use a systems biology approach to analyse a ciprofloxacin-selected multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium, L664.
The whole genome sequence and transcriptome of L664 were analysed. Site-directed mutagenesis to recreate each mutation was carried out, followed by phenotypic characterization and mutation frequency analysis. As a mutation in the TCA cycle was detected we tested the controversial hypothesis regarding the bacterial response to bactericidal antibiotics, put forward by Kohanski et al. (Cell 2007; 130: 797–810 and Mol Cell 2010; 37: 311–20), that exposure of bacteria to agents such as ciprofloxacin produces reactive oxygen species (ROS), which transiently increase the mutation rate giving rise to MDR bacteria.
L664 contained a mutation in ramR that conferred MDR. A mutation in tctA affected the TCA cycle and conferred the inability to grow on minimal agar. The virulence of L664 was not attenuated. Ciprofloxacin exposure produced ROS in L664 and SL1344 (tctA::aph), but it was reduced and occurred later. There were no significant differences in the rates of killing or mutations per generation to antibiotic resistance between the strains.
Whilst we confirm production of ROS in response to ciprofloxacin, we have no data to support the hypothesis that this leads to selection of MDR strains. Our results indicate that the mutations in tctA and glgA were random as they did not pre-exist in the parental strain, and that the mutation in tctA did not provide a survival advantage or disadvantage in the presence of antibiotic.
PMCID: PMC4125622  PMID: 22186876
TctA; RamR; efflux pumps; genome sequences

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