Genomic Characterization of a Large Outbreak of Legionella pneumophila Serogroup 1 Strains in Quebec City, 2012

During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire''s disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.     

Evidence for a Genetically Stable Strain of Campylobacter jejuni

The genetic stability of selected epidemiologically linked strains of Campylobacter jejuni during outbreak situations was investigated by using subtyping techniques. Strains isolated from geographically related chicken flock outbreaks in 1998 and from a human outbreak in 1981 were investigated. There was little similarity in the strains obtained from the different chicken flock outbreaks; however, the strains from each of three chicken outbreaks, including strains isolated from various environments, were identical as determined by fla typing, amplified fragment length polymorphism (AFLP) analysis, and pulsed-field gel electrophoresis, which confirmed the genetic stability of these strains during the short time courses of chicken flock outbreaks. The human outbreak samples were compared with strain 81116, which originated from the same outbreak but has since undergone innumerable laboratory passages. Two main AFLP profiles were recognized from this outbreak, which confirmed the serotyping results obtained at the time of the outbreak. The major type isolated from this outbreak (serotype P6:L6) was exemplified by strain 81116. Despite the long existence of strain 81116 as a laboratory strain, the AFLP profile of this strain was identical to the profiles of all the other historical P6:L6 strains from the outbreak, indicating that the genotype has remained stable for almost 20 years. Interestingly, the AFLP profiles of the P6:L6 group of strains from the human outbreak and the strains from one of the recent chicken outbreaks were also identical. This similarity suggests that some clones of C. jejuni remain genetically stable in completely different environments over long periods of time and considerable geographical distances.     

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

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.     

Comparative Genomic Analysis of Two Novel Sporadic Shiga Toxin-Producing Escherichia coli O104:H4 Strains Isolated 2011 in Germany

A large outbreak of gastrointestinal disease occurred in 2011 in Germany which resulted in almost 4000 patients with acute gastroenteritis or hemorrhagic colitis, 855 cases of a hemolytic uremic syndrome and 53 deaths. The pathogen was an uncommon, multiresistant Escherichia coli strain of serotype O104:H4 which expressed a Shiga toxin characteristic of enterohemorrhagic E. coli and in addition virulence factors common to enteroaggregative E. coli. During post-epidemic surveillance of Shiga toxin-producing E. coli (STEC) all but two of O104:H4 isolates were indistinguishable from the epidemic strain. Here we describe two novel STEC O104:H4 strains isolated in close spatiotemporal proximity to the outbreak which show a virulence gene panel, a Shiga toxin-mediated cytotoxicity towards Vero cells and aggregative adherence to Hep-2 cells comparable to the outbreak strain. They differ however both from the epidemic strain and from each other, by their antibiotic resistance phenotypes and some other features as determined by routine epidemiological subtyping methods. Whole genome sequencing of these two strains, of ten outbreak strain isolates originating from different time points of the outbreak and of one historical sporadic EHEC O104:H4 isolate was performed. Sequence analysis revealed a clear phylogenetic distance between the two variant strains and the outbreak strain finally identifying them as epidemiologically unrelated isolates from sporadic cases. These findings add to the knowledge about this emerging pathogen, illustrating a certain diversity within the bacterial core genome as well as loss and gain of accessory elements. Our results do also support the view that distinct new variants of STEC O104:H4 repeatedly might originate from yet unknown reservoirs, rather than that there would be a continuous diversification of a single epidemic strain established and circulating in Germany after the large outbreak in 2011.     

Genomic Characterization of Listeria monocytogenes Strains Involved in a Multistate Listeriosis Outbreak Associated with Cantaloupe in US

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.     

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak

Background

Waterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000.

Results

One of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains.

Conclusions

Our results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-768) contains supplementary material, which is available to authorized users.     

E. coli outbreak in a neonate intensive care unit in a general hospital in Mexico City

Nosocomial infections are a major cause of morbidity and mortality among neonates admitted to neonatal intensive care units (NICUs). The aim of this paper was to describe an outbreak of Escherichia coli among infants admitted to the NICU of the General Hospital “Dr. Manuel Gea Gonzalez” in May of 2008. The isolated E. coli strains were identified using standard biochemical methods. The susceptibilities of these strains were analysed by determining their minimal inhibitory concentrations. Following this, their molecular relationships to each other were assessed by pulsed field gel electrophoresis (PFGE) analysis and corroborated by serology. Twelve E. coli strains were isolated from blood, urine, or indwelling catheter samples from five cases of preterm infants within a 3-day period. Patients were admitted to the NICU of the general hospital and, during the outbreak, developed sepsis caused by E. coli. For four of the patients, the average age was 23 days, while one patient was a 3-month-old infant. Prior to sepsis, the infants had received assisted ventilation and hyperalimentation through a central venous catheter. Two profiles were observed by PFGE; profile A was identified as the outbreak’s cause and an outcome of cross-infection, while profile B showed genetic differences but serologically it was identified as part of the same serotype. We conclude that E. coli colonised the patients through horizontal transmission. A focal source of the microorganism in this outbreak was not identified, but cross-transmission through handling was the most probable route.     

Application of Multiple-Locus Variable Number Tandem Repeat Analysis to Identify Outbreak-Associated Neisseria meningitides Serogroup C Sequence Type 4821 in China

Neisseria meningitidis (N. meningitidis) serogroup C sequence type (ST)-4821 caused an outbreak in 2010 in Shandong province of China. Twenty-one non-outbreak-associated strains were isolated, along with twenty-eight N. meningitides serogroup C ST-4821 isolates. Therefore, it’s essential to identify and clarify characterization of the real outbreak-associated strains with a rapid method during an outbreak investigation. In this study, multiple-locus variable number tandem repeat analysis (MLVA) was applied to analyze 84 N. meningitidis strains, among which 58 were recovered from two outbreaks and 26 were sporadic isolates. Three MLVA schemes with different combination of VNTR loci were tested, and two of them were suitable for isolates from China: scheme 2 with six loci was found to separate ST into finer resolution, and scheme 3 with five loci can be used to identify outbreak-associated isolates from the same outbreak that caused by N. meningitidis serogroup C ST-4821.     

Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid,and Prophage Heterogeneity,Including Shiga Toxin Encoding Phage stx2

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.     

Characterization of Listeria monocytogenes Strains Involved in Invasive and Noninvasive Listeriosis Outbreaks by PCR-Based Fingerprinting Techniques

A total of 32 Listeria monocytogenes strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated L. monocytogenes food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.     

Pseudomonas syringae pv. actinidiae from Recent Outbreaks of Kiwifruit Bacterial Canker Belong to Different Clones That Originated in China

A recently emerged plant disease, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis), is caused by Pseudomonas syringae pv. actinidiae (PSA). The disease was first reported in China and Japan in the 1980s. A severe outbreak of PSA began in Italy in 2008 and has spread to other European countries. PSA was found in both New Zealand and Chile in 2010. To study the evolution of the pathogen and analyse the transmission of PSA between countries, genomes of strains from China and Japan (where the genus Actinidia is endemic), Italy, New Zealand and Chile were sequenced. The genomes of PSA strains are very similar. However, all strains from New Zealand share several single nucleotide polymorphisms (SNPs) that distinguish them from all other PSA strains. Similarly, all the PSA strains from the 2008 Italian outbreak form a distinct clonal group and those from Chile form a third group. In addition to the rare SNPs present in the core genomes, there is abundant genetic diversity in a genomic island that is part of the accessory genome. The island from several Chinese strains is almost identical to the island present in the New Zealand strains. The island from a different Chinese strain is identical to the island present in the strains from the recent Italian outbreak. The Chilean strains of PSA carry a third variant of this island. These genomic islands are integrative conjugative elements (ICEs). Sequencing of these ICEs provides evidence of three recent horizontal transmissions of ICE from other strains of Pseudomonas syringae to PSA. The analyses of the core genome SNPs and the ICEs, combined with disease history, all support the hypothesis of an independent Chinese origin for both the Italian and the New Zealand outbreaks and suggest the Chilean strains also originate from China.     

Multilocus Sequence Analysis for Leishmania braziliensis Outbreak Investigation

With the emergence of leishmaniasis in new regions around the world, molecular epidemiological methods with adequate discriminatory power, reproducibility, high throughput and inter-laboratory comparability are needed for outbreak investigation of this complex parasitic disease. As multilocus sequence analysis (MLSA) has been projected as the future gold standard technique for Leishmania species characterization, we propose a MLSA panel of six housekeeping gene loci (6pgd, mpi, icd, hsp70, mdhmt, mdhnc) for investigating intraspecific genetic variation of L. (Viannia) braziliensis strains and compare the resulting genetic clusters with several epidemiological factors relevant to outbreak investigation. The recent outbreak of cutaneous leishmaniasis caused by L. (V.) braziliensis in the southern Brazilian state of Santa Catarina is used to demonstrate the applicability of this technique. Sequenced fragments from six genetic markers from 86 L. (V.) braziliensis strains from twelve Brazilian states, including 33 strains from Santa Catarina, were used to determine clonal complexes, genetic structure, and phylogenic networks. Associations between genetic clusters and networks with epidemiological characteristics of patients were investigated. MLSA revealed epidemiological patterns among L. (V.) braziliensis strains, even identifying strains from imported cases among the Santa Catarina strains that presented extensive homogeneity. Evidence presented here has demonstrated MLSA possesses adequate discriminatory power for outbreak investigation, as well as other potential uses in the molecular epidemiology of leishmaniasis.     

Two-Step Source Tracing Strategy of Yersinia pestis and Its Historical Epidemiology in a Specific Region

Source tracing of pathogens is critical for the control and prevention of infectious diseases. Genome sequencing by high throughput technologies is currently feasible and popular, leading to the burst of deciphered bacterial genome sequences. Utilizing the flooding genomic data for source tracing of pathogens in outbreaks is promising, and challenging as well. Here, we employed Yersinia pestis genomes from a plague outbreak at Xinghai county of China in 2009 as an example, to develop a simple two-step strategy for rapid source tracing of the outbreak. The first step was to define the phylogenetic position of the outbreak strains in a whole species tree, and the next step was to provide a detailed relationship across the outbreak strains and their suspected relatives. Through this strategy, we observed that the Xinghai plague outbreak was caused by Y. pestis that circulated in the local plague focus, where the majority of historical plague epidemics in the Qinghai-Tibet Plateau may originate from. The analytical strategy developed here will be of great help in fighting against the outbreaks of emerging infectious diseases, by pinpointing the source of pathogens rapidly with genomic epidemiological data and microbial forensics information.     

Whole-Genome Sequencing for the Investigation of a Hospital Outbreak of MRSA in China

Staphylococcus aureus is a globally disseminated drug-resistant bacterial species. It remains a leading cause of hospital-acquired infection, primarily among immunocompromised patients. In 2012, the Affiliated People’s Hospital of Jiangsu University experienced a putative outbreak of methicillin-resistant S. aureus (MRSA) that affected 12 patients in the Neurosurgery Department. In this study, whole-genome sequencing (WGS) was used to gain insight into the epidemiology of the outbreak caused by MRSA, and traditional bacterial genotyping approaches were also applied to provide supportive evidence for WGS. We sequenced the DNA from 6 isolates associated with the outbreak. Phylogenetic analysis was constructed by comparing single-nucleotide polymorphisms (SNPs) in the core genome of 6 isolates in the present study and another 3 referenced isolates from GenBank. Of the 6 MRSA sequences in the current study, 5 belonged to the same group, clustering with T0131, while the other one clustered closely with TW20. All of the isolates were identified as ST239-SCCmecIII clones. Whole-genome analysis revealed that four of the outbreak isolates were more tightly clustered into a group and SA13002 together with SA13009 were distinct from the outbreak strains, which were considered non-outbreak strains. Based on the sequencing results, the antibiotic-resistance gene status (present or absent) was almost perfectly concordant with the results of phenotypic susceptibility testing. Various toxin genes were also analyzed successfully. Our analysis demonstrates that using traditional molecular methods and WGS can facilitate the identification of outbreaks and help to control nosocomial transmission.     

Molecular Analysis of Pseudomonas aeruginosa: Epidemiological Investigation of Mastitis Outbreaks in Irish Dairy Herds

Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.     

Draft Genome Sequences of 21 Salmonella enterica Serovar Enteritidis Strains

Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.     

Genetic Markers Unique to Listeria monocytogenes Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.     

Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India

We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139–147 contigs. Average sequencing depth for all four genomes was 21x.     

Two Novel EHEC/EAEC Hybrid Strains Isolated from Human Infections

The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H⁻, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H⁻, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.     

Using Neisseria meningitidis genomic diversity to inform outbreak strain identification

Meningococcal disease is a life-threatening illness caused by the human-restricted bacterium Neisseria meningitidis. Outbreaks in the USA involve at least two cases in an organization or community caused by the same serogroup within three months. Genome comparisons, including phylogenetic analysis and quantification of genome distances can provide confirmatory evidence of pathogen transmission during an outbreak. Interpreting genome distances depends on understanding their distribution both among isolates from outbreaks and among those not from outbreaks. Here, we identify outbreak strains based on phylogenetic relationships among 141 N. meningitidis isolates collected from 28 outbreaks in the USA during 2010–2017 and 1516 non-outbreak isolates collected through contemporaneous meningococcal surveillance. We show that genome distance thresholds based on the maximum SNPs and allele distances among isolates in the phylogenetically defined outbreak strains are sufficient to separate most pairs of non-outbreak isolates into separate strains. Non-outbreak isolate pairs that could not be distinguished from each other based on genetic distances were concentrated in the clonal complexes CC11, CC103, and CC32. Within each of these clonal complexes, phylodynamic analysis identified a group of isolates with extremely low diversity, collected over several years and multiple states. Clusters of isolates with low genetic diversity could indicate increased pathogen transmission, potentially resulting in local outbreaks or nationwide clonal expansions.