Bacterial resistance to antibiotics is a pressing health issue around the world, not only in heal... more Bacterial resistance to antibiotics is a pressing health issue around the world, not only in health care settings but also in the community and environment, particularly in crowded urban populations. The aim of our work was to characterize the microbial populations in sewage and the spread of antibiotic resistance within New York City (NYC). Here, we investigated the structure of the microbiome and the prevalence of antibiotic resistance genes in raw sewage samples collected from the fourteen NYC Department of Environmental Protection wastewater treatment plants, distributed across the five NYC boroughs. Sewage, a direct output of anthropogenic activity and a reservoir of microbes, provides an ecological niche to examine the spread of antibiotic resistance. Taxonomic diversity analysis revealed a largely similar and stable bacterial population structure across all the samples, which was found to be similar over three time points in an annual cycle, as well as in the five NYC boroughs. All samples were positive for the presence of the seven antibiotic resistance genes tested, based on real-time quantitative PCR assays, with higher levels observed for tetracycline resistance genes at all time points. For five of the seven genes, abundances were significantly higher in May than in February and August. This study provides characteristics of the NYC sewage resistome in the context of the overall bacterial populations.
To the Editor: To overcome various limitations of phenotyping and 16S rDNA sequence analysis of C... more To the Editor: To overcome various limitations of phenotyping and 16S rDNA sequence analysis of Cronobacter bacteria, we have established a comprehensive multilocus sequence typing (MLST) scheme as an open access database resource (www. pubMLST. org/cronobacter)(1). The scheme is based on 7 housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3,036 nt concatenated length) and has been used to study the diversity of the Cronobacter genus and new Cronobacter species (2–4).
Abstract A 7-loci (3,036 nt) multilocus sequence typing scheme was applied to 41 clinical isolate... more Abstract A 7-loci (3,036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Cronobacter sakazakii. Half (20/41) of the C. sakazakii strains were sequence type (ST) 4, and 9/12 meningitis isolates were ST4. C. sakazakii ST4 appears to be a highly stable clone with a high propensity for neonatal meningitis.
Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting a... more Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognised route of infection being the consumption of contaminated infant formula. As a recently recognised bacterial pathogen of considerable importance and regulatory control, appropriate detection and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognised genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health.
The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tr... more The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tract infections (UTIs) in clinical laboratories does not report bacteria isolated from samples containing three or more different bacterial species. As a result many UTIs go unreported and untreated, particularly in elderly patients, where polymicrobial UTI samples are especially prevalent. This study reports the presence of the major uropathogenic species in mixed culture urine samples from elderly patients, and of resistance to front-line antibiotics, with potentially increased levels of resistance to ciprofloxacin and trimethoprim. Most importantly, the study highlights that Escherichia coli present in polymicrobial UTI samples are statistically more invasive (P<0.001) in in vitro epithelial cell infection assays than those isolated from monomicrobial culture samples. In summary, the results of this study suggest that the current diagnostic standard procedure for polymicrobial UTI samples needs to be reassessed, and that E. coli present in polymicrobial UTI samples may pose an increased risk to human health.
Members of the Cronobacter genus (formerly Enterobacter sakazakii) have become associated with ne... more Members of the Cronobacter genus (formerly Enterobacter sakazakii) have become associated with neonatal infections and in particular contaminated reconstituted infant formula. However this is only one perspective of the organism since the majority of infections are in the adult population, and the organism has been isolated from the enteral feeding tubes of neonates on non-formula diets. In recent years methods of detection from food and environmental sources have improved, though accurate identification has been problematic. The need for robust identification is essential in order to implement recent Codex Alimentarius Commission (2008) and related microbiological criteria for powdered infant formula (PIF; intended target age 0-6 months). Genomic analysis of emergent pathogens is of considerable advantage in both improving detection methods, and understanding the evolution of virulence. One ecosystem for Cronobacter is on plant material which may enable the organism to resist desiccation, adhere to surfaces, and resist some antimicrobial agents. These traits may also confer survival mechanisms of relevance in food manufacturing and also virulence mechanisms.
A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii(... more A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii(1330T), two previously identified as Cronobacter genomospecies 1 (strain NCTC 9529T and strain 731), and two as Cronobacter turicensis (strain 96 and 1435) was carried out using a polyphasic approach. The analysis included a phenotypic characterization, sequencing of the 16S rRNA and a Multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). The 16S rRNA and MLSA analyses showed strain 1330T, isolated from spiced meat purchased in Slovakia, to form an independent phylogenetic line. Cronobacter dublinensis was the closest neighbour species on the basis of the MLSA. DNA-DNA reassociation experiments and phenotypic analysis revealed that strain 1330T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed, type strain 1330T =CECT 7863T, =LMG 26250T). The 4 bacterial strains NCTC 9529T, 731, 96, and 1435, isolated from water, a leg infection and two food ingredients; onion powder and rye flour, respectively, showed on the MLSA phylogenetic tree to cluster together within an independent phylogenetic line, with Cronobacter turicensis as the closest species. The DNA-DNA hybridization data and the phenotypic characterization confirmed that these strains represented a novel species, for which the name Cronobacter universalis sp. nov. is proposed with type strain NCTC 9529T =CECT 7864T,=LMG 26249T.
A 7 loci (3036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Crono... more A 7 loci (3036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Cronobacter sakazakii. Half (20/41) of the C. sakazakii strains were ST4, and 9/12 meningitis isolates were ST4. C. sakazakii ST4 appears to be a highly stable clone with a high propensity for neonatal meningitis.
Background
Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known ... more Background Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of Cronobacter do not differentiate between species, necessitating time consuming 16S rDNA gene sequencing or multilocus sequence typing (MLST). The organism is ubiquitous, being found in the environment and in a wide range of foods, although there is variation in pathogenicity between Cronobacter isolates and between species. Therefore to be able to differentiate between the pathogenic and non-pathogenic strains is of interest to the food industry and regulators.
Results Here we report the use of Expectation Maximization clustering to categorise 98 strains of Cronobacter as pathogenic or non-pathogenic based on biochemical test results from standard diagnostic test kits. Pathogenicity of a strain was postulated on the basis of either pathogenic symptoms associated with strain source or corresponding MLST sequence types, allowing the clusters to be labelled as containing either pathogenic or non-pathogenic strains. The resulting clusters gave good differentiation of strains into pathogenic and non-pathogenic groups, corresponding well to isolate source and MLST sequence type. The results also revealed a potential association between pathogenicity and inositol fermentation. An investigation of the genomes of Cronobacter sakazakii and C. turicensis revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of Cronobacter.
Conclusions We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of Cronobacter. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of Cronobacter species.
Bacterial resistance to antibiotics is a pressing health issue around the world, not only in heal... more Bacterial resistance to antibiotics is a pressing health issue around the world, not only in health care settings but also in the community and environment, particularly in crowded urban populations. The aim of our work was to characterize the microbial populations in sewage and the spread of antibiotic resistance within New York City (NYC). Here, we investigated the structure of the microbiome and the prevalence of antibiotic resistance genes in raw sewage samples collected from the fourteen NYC Department of Environmental Protection wastewater treatment plants, distributed across the five NYC boroughs. Sewage, a direct output of anthropogenic activity and a reservoir of microbes, provides an ecological niche to examine the spread of antibiotic resistance. Taxonomic diversity analysis revealed a largely similar and stable bacterial population structure across all the samples, which was found to be similar over three time points in an annual cycle, as well as in the five NYC boroughs. All samples were positive for the presence of the seven antibiotic resistance genes tested, based on real-time quantitative PCR assays, with higher levels observed for tetracycline resistance genes at all time points. For five of the seven genes, abundances were significantly higher in May than in February and August. This study provides characteristics of the NYC sewage resistome in the context of the overall bacterial populations.
To the Editor: To overcome various limitations of phenotyping and 16S rDNA sequence analysis of C... more To the Editor: To overcome various limitations of phenotyping and 16S rDNA sequence analysis of Cronobacter bacteria, we have established a comprehensive multilocus sequence typing (MLST) scheme as an open access database resource (www. pubMLST. org/cronobacter)(1). The scheme is based on 7 housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3,036 nt concatenated length) and has been used to study the diversity of the Cronobacter genus and new Cronobacter species (2–4).
Abstract A 7-loci (3,036 nt) multilocus sequence typing scheme was applied to 41 clinical isolate... more Abstract A 7-loci (3,036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Cronobacter sakazakii. Half (20/41) of the C. sakazakii strains were sequence type (ST) 4, and 9/12 meningitis isolates were ST4. C. sakazakii ST4 appears to be a highly stable clone with a high propensity for neonatal meningitis.
Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting a... more Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognised route of infection being the consumption of contaminated infant formula. As a recently recognised bacterial pathogen of considerable importance and regulatory control, appropriate detection and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognised genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health.
The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tr... more The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tract infections (UTIs) in clinical laboratories does not report bacteria isolated from samples containing three or more different bacterial species. As a result many UTIs go unreported and untreated, particularly in elderly patients, where polymicrobial UTI samples are especially prevalent. This study reports the presence of the major uropathogenic species in mixed culture urine samples from elderly patients, and of resistance to front-line antibiotics, with potentially increased levels of resistance to ciprofloxacin and trimethoprim. Most importantly, the study highlights that Escherichia coli present in polymicrobial UTI samples are statistically more invasive (P<0.001) in in vitro epithelial cell infection assays than those isolated from monomicrobial culture samples. In summary, the results of this study suggest that the current diagnostic standard procedure for polymicrobial UTI samples needs to be reassessed, and that E. coli present in polymicrobial UTI samples may pose an increased risk to human health.
Members of the Cronobacter genus (formerly Enterobacter sakazakii) have become associated with ne... more Members of the Cronobacter genus (formerly Enterobacter sakazakii) have become associated with neonatal infections and in particular contaminated reconstituted infant formula. However this is only one perspective of the organism since the majority of infections are in the adult population, and the organism has been isolated from the enteral feeding tubes of neonates on non-formula diets. In recent years methods of detection from food and environmental sources have improved, though accurate identification has been problematic. The need for robust identification is essential in order to implement recent Codex Alimentarius Commission (2008) and related microbiological criteria for powdered infant formula (PIF; intended target age 0-6 months). Genomic analysis of emergent pathogens is of considerable advantage in both improving detection methods, and understanding the evolution of virulence. One ecosystem for Cronobacter is on plant material which may enable the organism to resist desiccation, adhere to surfaces, and resist some antimicrobial agents. These traits may also confer survival mechanisms of relevance in food manufacturing and also virulence mechanisms.
A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii(... more A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii(1330T), two previously identified as Cronobacter genomospecies 1 (strain NCTC 9529T and strain 731), and two as Cronobacter turicensis (strain 96 and 1435) was carried out using a polyphasic approach. The analysis included a phenotypic characterization, sequencing of the 16S rRNA and a Multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). The 16S rRNA and MLSA analyses showed strain 1330T, isolated from spiced meat purchased in Slovakia, to form an independent phylogenetic line. Cronobacter dublinensis was the closest neighbour species on the basis of the MLSA. DNA-DNA reassociation experiments and phenotypic analysis revealed that strain 1330T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed, type strain 1330T =CECT 7863T, =LMG 26250T). The 4 bacterial strains NCTC 9529T, 731, 96, and 1435, isolated from water, a leg infection and two food ingredients; onion powder and rye flour, respectively, showed on the MLSA phylogenetic tree to cluster together within an independent phylogenetic line, with Cronobacter turicensis as the closest species. The DNA-DNA hybridization data and the phenotypic characterization confirmed that these strains represented a novel species, for which the name Cronobacter universalis sp. nov. is proposed with type strain NCTC 9529T =CECT 7864T,=LMG 26249T.
A 7 loci (3036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Crono... more A 7 loci (3036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Cronobacter sakazakii. Half (20/41) of the C. sakazakii strains were ST4, and 9/12 meningitis isolates were ST4. C. sakazakii ST4 appears to be a highly stable clone with a high propensity for neonatal meningitis.
Background
Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known ... more Background Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of Cronobacter do not differentiate between species, necessitating time consuming 16S rDNA gene sequencing or multilocus sequence typing (MLST). The organism is ubiquitous, being found in the environment and in a wide range of foods, although there is variation in pathogenicity between Cronobacter isolates and between species. Therefore to be able to differentiate between the pathogenic and non-pathogenic strains is of interest to the food industry and regulators.
Results Here we report the use of Expectation Maximization clustering to categorise 98 strains of Cronobacter as pathogenic or non-pathogenic based on biochemical test results from standard diagnostic test kits. Pathogenicity of a strain was postulated on the basis of either pathogenic symptoms associated with strain source or corresponding MLST sequence types, allowing the clusters to be labelled as containing either pathogenic or non-pathogenic strains. The resulting clusters gave good differentiation of strains into pathogenic and non-pathogenic groups, corresponding well to isolate source and MLST sequence type. The results also revealed a potential association between pathogenicity and inositol fermentation. An investigation of the genomes of Cronobacter sakazakii and C. turicensis revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of Cronobacter.
Conclusions We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of Cronobacter. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of Cronobacter species.
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Papers by Susan Joseph
Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of Cronobacter do not differentiate between species, necessitating time consuming 16S rDNA gene sequencing or multilocus sequence typing (MLST). The organism is ubiquitous, being found in the environment and in a wide range of foods, although there is variation in pathogenicity between Cronobacter isolates and between species. Therefore to be able to differentiate between the pathogenic and non-pathogenic strains is of interest to the food industry and regulators.
Results
Here we report the use of Expectation Maximization clustering to categorise 98 strains of Cronobacter as pathogenic or non-pathogenic based on biochemical test results from standard diagnostic test kits. Pathogenicity of a strain was postulated on the basis of either pathogenic symptoms associated with strain source or corresponding MLST sequence types, allowing the clusters to be labelled as containing either pathogenic or non-pathogenic strains. The resulting clusters gave good differentiation of strains into pathogenic and non-pathogenic groups, corresponding well to isolate source and MLST sequence type. The results also revealed a potential association between pathogenicity and inositol fermentation. An investigation of the genomes of Cronobacter sakazakii and C. turicensis revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of Cronobacter.
Conclusions
We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of Cronobacter. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of Cronobacter species.
Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of Cronobacter do not differentiate between species, necessitating time consuming 16S rDNA gene sequencing or multilocus sequence typing (MLST). The organism is ubiquitous, being found in the environment and in a wide range of foods, although there is variation in pathogenicity between Cronobacter isolates and between species. Therefore to be able to differentiate between the pathogenic and non-pathogenic strains is of interest to the food industry and regulators.
Results
Here we report the use of Expectation Maximization clustering to categorise 98 strains of Cronobacter as pathogenic or non-pathogenic based on biochemical test results from standard diagnostic test kits. Pathogenicity of a strain was postulated on the basis of either pathogenic symptoms associated with strain source or corresponding MLST sequence types, allowing the clusters to be labelled as containing either pathogenic or non-pathogenic strains. The resulting clusters gave good differentiation of strains into pathogenic and non-pathogenic groups, corresponding well to isolate source and MLST sequence type. The results also revealed a potential association between pathogenicity and inositol fermentation. An investigation of the genomes of Cronobacter sakazakii and C. turicensis revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of Cronobacter.
Conclusions
We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of Cronobacter. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of Cronobacter species.