Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

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Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

From international tourists to war-displaced refugees, more people are on the move than ever before. This provides the opportunity for a variety of antimicrobial-resistant bacteria to be carried from one geographic location to another. The Enterobacteriaceae are among the most important causes of serious hospital-acquired and community-onset bacterial infections in humans, and resistance to antimicrobial agents in these bacteria has become an increasingly relevant problem. International travel and tourism are important modes for the acquisition and spread of antimicrobial-resistant Enterobacteriaceae, especially CTX-M-producing Escherichia coli. Infections with KPC-, VIM-, OXA-48- and NDM-producing Enterobacteriaceae in developed countries have been associated with visiting and being hospitalized in endemic areas such as the USA, Greece and Israel for KPCs, Greece for VIMs, Turkey for OXA-48, and the Indian subcontinent for NDMs. To combat the spread of antimicrobial-resistant Enterobacteriaceae, the French Healthcare Safety Advisory Committee recently issued national recommendations for screening and contact isolation precautions for patients transferred from, or hospitalized outside, France. For effective public and patient health interventions, it is important to understand the role of international travel in the spread of antimicrobial-resistant Enterobacteriaceae. We urgently need well-designed studies to evaluate the transmission potential and risks for colonization and infections due to multiresistant Enterobacteriaceae in travellers who have recently visited or have been hospitalized in endemic areas. The emergence of CTX-M-, KPC- and NDM-producing bacteria is a good example of the role that globalization plays in the rapid dissemination of new antibiotic resistance mechanisms.

The role of international travel in the worldwide spread of multiresistant Enterobacteriaceae

For more than 80 years, subtyping of Salmonella enterica has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of Salmonella in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different Salmonella enterica subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping Salmonella strains at a deeper level.

Methodologies for Salmonella enterica subsp. enterica subtyping: gold standards and alternatives.

Background: In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients. Methods: We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali. Principal Findings: We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.

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Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 104 CFU mL−1 or 105 CFU mL−1 for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R2) of 0.916–0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

/pdf/rapid-multiplex-detection-of-10-foodborne-pathogens-with-an-q7oqcszs1d.pdf

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay

A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subsp. enterica encountered in Washington State and the United States in general. Six genetic loci from S. enterica serovar Typhimurium and four from S. enterica serovar Typhi were used to create an assay consisting of two five-plex PCRs. The assays gave reproducible results with 30 different serotypes that represent the most common clinical isolates of S. enterica subsp. enterica. Of these, 22 serotypes gave unique amplification patterns compared with each other and the other 8 serotypes were grouped into four pairs. These were further resolved by two additional PCRs. We compared the data from PCR serotyping with conventional serotyping and found that PCR serotyping was nearly as discriminatory as conventional serotyping was. The results from a blind test screening 111 clinical isolates revealed that 97% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 h and, in conjunction with pulsed-field gel electrophoresis, forms a very robust test method for the molecular subtyping of Salmonella enterica subsp. enterica.

Multiplex PCR-Based Method for Identification of Common Clinical Serotypes of Salmonella enterica subsp. enterica

Salmonella enterica is a leading cause of food-borne illness worldwide and is also a major cause of morbidity and mortality in domestic and wild animals. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and nonhuman serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR while incorporating a 6-carboxyfluorescein-labeled universal primer to fluorescently label all amplicons. The fluorescently labeled PCR products were separated using capillary electrophoresis, and a Salmonella multiplex assay for rapid typing (SMART) code was generated for each isolate, based upon the presence or absence of PCR products generated from each target gene. Seven hundred fifty-one blind clinical isolates of Salmonella from Washington State, collected in 2007 and previously serotyped via antisera, were screened with the assay. A total of 89.6% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously determined serotype, while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to that of conventional serotyping, but at a fraction of both the cost and time required per test.

High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis

To the Editor: The CTX-M–type β-lactamases are non-TEM and non-SHV plasmid-mediated, class A, extended-spectrum β-lactamases (ESBLs). The CTX-M–type β-lactamases have recently emerged as the most common type of ESBLs, with a global distribution (1). In contrast, the CTX-M–type ESBLs are rarely reported in the United States and have not been identified in pathogens isolated from infected patients with gastroenteritis.

We screened 637 Salmonella and 126 Shigella isolates, collected in the state of Washington during 2003–2004, for CTX-M–type β-lactamases. Of these, 60 Salmonella isolates that exhibited an ESBL phenotype were further characterized by PCR for TEV, SHV, CTM-X, and CMY. All were positive for the CMY-2 or TEM-1 β-lactam genes. One Shigella sonnei isolate (WA7593), cultured from a fecal specimen in August 2004, tested positive with an ESBL confirmatory disk diffusion panel (ceftazidime 24 mm, ceftazidime/clavulanate 32 mm, cefotaxime 14 mm, and cefotaxime/clavulanate 34 mm; [2]). The patient had recently traveled to Pakistan and likely became ill there and returned to the United States while still sick. The transfer of extended-spectrum cephalosporin resistance was tested by conjugation to Escherichia coli J53 aziR (3). The MIC for S. sonnei WA7593 and its transconjugant, WA7593TC1, were tested by using the E-test (AB Biodisk, Solna, Sweden). Both strains were resistant to cefotaxime and susceptible to ceftazidime and showed almost the same antimicrobial susceptibility patterns as β-lactam antimicrobial drugs (Table).



Table

MICs of antimicrobial drugs for Shigella sonnei clinical isolate WA7593 and its transconjugant WA7593TC1



The type of ESBL produced by these strains was determined by using PCR specific for TEM and CTX-M (4,5). Both strains were PCR positive for TEM and CTX-M. The TEM type PCR products were then sequenced and identified as TEM-1; no variation was found on the promoter region of blaTEM-1. The entire sequence of bla CTX-M from WA7593 was then sequenced (1), and the product showed 100% homology with blaCTX-M-15 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY960984","term_id":"63029819","term_text":"AY960984"}}AY960984). The mobile element associated with the transfer of blaCTX-M-15 was investigated by sequencing the flanking regions. PCRs were performed with primers from the internal regions of bla CTX-M gene and primers for insertion sequences ISEcp1 and IS903 (4,5). Positive PCR products were obtained with primers ISEcp1F and CTX2 (943 bp); no amplified product was produced with primers CTX1 and IS903R. Sequencing of a 943-bp amplicon showed that blaCTX-M15 was flanked upstream by an ISEcp1-like element.

The presence of an integron in S. sonnei WA7593 and WA7593TC1 was investigated by using integron-specific primers hep35 and hep36 (2). Only S. sonnei WA7593 produced a PCR product. This finding suggests that the transmission of blaCTX-M15 is not by integron-mediated transfer. A further 162 Shigella spp. and 260 Salmonella spp. isolated from 2003 through 2005 were also screened for ESBL production; no further isolates were identified.

The presence of a CTX-M–type, ESBL-producing isolate is rarely reported in the United States. The only other reference was from a multistate study in 2001–2002 that identified CTX-M type from E. coli isolates from urine, sputum, and blood (6). No further reports about CTX-M–producing organisms have been disseminated. Our investigation suggests that CTX-M–type ESBLs may spread throughout the United States through infected travelers. This finding is notable because S. sonnei is a common enteric pathogen. Our results further emphasize that travelers from others parts of the world can introduce highly mobile and clinically important resistance mechanisms into the community. The spread of CTX-M ESBLs may be faster and more widespread than previously thought; therefore, CTX-M type should be taken seriously as a surveillance target in the United States, especially in patients with a history of travel outside North America.

CTX-M Extended-spectrum β-Lactamases, Washington State

Functional analysis of TaPDI genes on storage protein accumulation by CRISPR/Cas9 edited wheat mutants

Seeking alternatives to sensory which can be performed in non-laboratory field environments has been a recent emphasis in regulatory assessment of seafood decomposition.  To this end, an international sampling effort produced samples of 23 different seafood products, which were subjected to controlled decomposition of varying levels on ice. This produced samples of each product for a wide range of decomposition states, and these were scored by a sensory expert on a standard 1-100 scale. Samples were then analyzed by a novel technique involving headspace analysis and compact mass spectrometry. The mass spectrometry data was used to create computer models, guided by the sensory data, to generate a calculated score analogous to a sensory score. Models based on a training set of samples were then used to calculate scores for a test set, and accuracy was assessed by comparing these calculated scores to original sensory data. Calculated scores agreed with sensory findings with respect to overall decomposition state for 96.3% of samples (n=547), with seven false positive (1.3%) and 13 false negative (2.4%) findings. Reproducibility was also assessed via triplicate analysis on separate days for low, middle, and high decomposition states. All sample ranges for these replicates were within 20 points, with 89% (n=66) within 15 points, 73% within 10, and 35% within five. Given the performance of the method, along with the small form factor and manageable operation requirements of the instrument as well as simplicity of sample preparation, this appears to offer a potentially useful technique for field testing of seafood decomposition.

Jinxin Hu

Papers

Multiplex PCR-Based Method for Identification of Common Clinical Serotypes of Salmonella enterica subsp. enterica

High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis

CTX-M Extended-spectrum β-Lactamases, Washington State

Functional analysis of TaPDI genes on storage protein accumulation by CRISPR/Cas9 edited wheat mutants

Rapid, field-deployable method for determination of seafood decomposition by compact mass spectrometry