Correction: Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica.

[This corrects the article DOI: 10.1371/journal.ppat.1002776.].

Multilocus sequence typing as a replacement for serotyping in Salmonella enterica.

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.

Infection biology of Salmonella enterica.

Salmonella enterica is the leading cause of bacterial foodborne illness in the USA, with an estimated 95% of salmonellosis cases due to the consumption of contaminated food products. Salmonella can cause several different disease syndromes, with the most common being gastroenteritis, followed by bacteremia and typhoid fever. Among the over 2,600 currently identified serotypes/serovars, some are mostly host-restricted and host-adapted, while the majority of serotypes can infect a broader range of host species and are associated with causing both livestock and human disease. Salmonella serotypes and strains within serovars can vary considerably in the severity of disease that may result from infection, with some serovars that are more highly associated with invasive disease in humans, while others predominantly cause mild gastroenteritis. These observed clinical differences may be caused by the genetic make-up and diversity of the serovars. Salmonella virulence systems are very complex containing several virulence-associated genes with different functions that contribute to its pathogenicity. The different clinical syndromes are associated with unique groups of virulence genes, and strains often differ in the array of virulence traits they display. On the chromosome, virulence genes are often clustered in regions known as Salmonella pathogenicity islands (SPIs), which are scattered throughout different Salmonella genomes and encode factors essential for adhesion, invasion, survival, and replication within the host. Plasmids can also carry various genes that contribute to Salmonella pathogenicity. For example, strains from several serovars associated with significant human disease, including Choleraesuis, Dublin, Enteritidis, Newport, and Typhimurium, can carry virulence plasmids with genes contributing to attachment, immune system evasion, and other roles. The goal of this comprehensive review is to provide key information on the Salmonella virulence, including the contributions of genes encoded in SPIs and plasmids during Salmonella pathogenesis.

Evolution and population structure of Salmonella enterica serovar Newport.

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.

Antimicrobial resistance, virulence, and genotypic profile comparison of Campylobacter jejuni and Campylobacter coli isolated from humans and retail meats.

A total of 360 spatially and temporally related Campylobacter isolates, including 168 from clinical human cases (Campylobacter jejuni n = 148; Campylobacter coli n = 20) and 192 from retail meats (C. jejuni n = 114; C. coli n = 78), were analyzed for antimicrobial susceptibilities, virulence, and genotypic profiles. Ciprofloxacin-resistant C. jejuni was observed in 13.5% and 19% of the isolates from humans and retail chicken breasts, respectively. Antimicrobial resistance to ciprofloxacin and erythromycin was detected in C. coli isolates recovered from 29% and 16.6% of retail meats and 15% and 5% humans, respectively. Overall, virulence determinants were more prevalent in Campylobacter isolates recovered from retail meats than from humans. C. jejuni isolates from humans were significantly associated with the rakR, dnaJ, and pld genes, whereas C. coli isolates from retail meats were associated with the dnaJ, pld, and virB11 virulence genes. Genotyping of 262 C. jejuni isolates using pulsed-field gel electrophoresis revealed a total of 186 unique SmaI patterns, with 14% of patterns composed of isolates recovered from retail meats and ill humans. All unique groups with indistinguishable SmaI patterns were further analyzed by a second restriction enzyme (KpnI), which revealed limited overlap between isolates from different sources. Significant association between doxycycline-resistant C. jejuni strains recovered from humans and different virulence genes (e.g., cdtB) was identified at the statistical level but not at the genotypic level. In conclusion, significant differences observed in the distribution of antimicrobial resistance profiles, virulence determinants, and genotypic diversity among C. jejuni and C. coli isolates indicate that there are sources other than retail meats that may also contribute to human Campylobacter infections.

Antimicrobial Drug Resistance in Fish Pathogens.

Major concerns surround the use of antimicrobial agents in farm-raised fish, including the potential impacts these uses may have on the development of antimicrobial-resistant pathogens in fish and the aquatic environment. Currently, some antimicrobial agents commonly used in aquaculture are only partially effective against select fish pathogens due to the emergence of resistant bacteria. Although reports of ineffectiveness in aquaculture due to resistant pathogens are scarce in the literature, some have reported mass mortalities in Penaeus monodon larvae caused by Vibrio harveyi resistant to trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, and streptomycin. Genetic determinants of antimicrobial resistance have been described in aquaculture environments and are commonly found on mobile genetic elements which are recognized as the primary source of antimicrobial resistance for important fish pathogens. Indeed, resistance genes have been found on transferable plasmids and integrons in pathogenic bacterial species in the genera Aeromonas, Yersinia, Photobacterium, Edwardsiella, and Vibrio. Class 1 integrons and IncA/C plasmids have been widely identified in important fish pathogens (Aeromonas spp., Yersinia spp., Photobacterium spp., Edwardsiella spp., and Vibrio spp.) and are thought to play a major role in the transmission of antimicrobial resistance determinants in the aquatic environment. The identification of plasmids in terrestrial pathogens (Salmonella enterica serotypes, Escherichia coli, and others) which have considerable homology to plasmid backbone DNA from aquatic pathogens suggests that the plasmid profiles of fish pathogens are extremely plastic and mobile and constitute a considerable reservoir for antimicrobial resistance genes for pathogens in diverse environments.

Effects of low concentrations of erythromycin, penicillin, and virginiamycin on bacterial resistance development in vitro.

Distillers grains are co-products of the corn ethanol industry widely used in animal feed. We examined the effects of erythromycin, penicillin, and virginiamycin at low concentrations reflective of those detected in distillers grains on bacterial resistance selection. At 0.1 µg/ml erythromycin, macrolide-resistant mutants were induced in one Campylobacter coli and one Enterococcus faecium strain, while these strains plus three additional C. coli, one additional E. faecium, and one C. jejuni also developed resistance when exposed to 0.25 µg/ml erythromycin. At 0.5 µg/ml erythromycin, a total of eight strains (four Campylobacter and four Enterococcus) obtained macrolide-resistant mutants, including two strains from each genus that were not selected at lower erythromycin concentrations. For penicillin, three of five E. faecium strains but none of five Enterococcus faecalis strains consistently developed resistance at all three selection concentrations. Virginiamycin at two M1:S1 ratios did not induce resistance development in four out of five E. faecium strains; however, increased resistance was observed in the fifth one under 0.25 and 0.5 µg/ml virginiamycin selections. Although not yet tested in vivo, these findings suggest a potential risk of stimulating bacterial resistance development in the animal gut when distillers grains containing certain antibiotic residues are used in animal feed.

Poly I:C induces an antiviral state against Ictalurid Herpesvirus 1 and Mx1 transcription in the channel catfish (Ictalurus punctatus).

In vivo studies were carried out to investigate the protective effect of the interferon inducer poly I:C against channel catfish virus (CCV). Channel catfish were stimulated by intraperitoneal injection of 50 microg of poly I:C or PBS at various days prior to immersion challenge with CCV. Mortality in the poly I:C group was significantly reduced from 70% to 3% at day 1 compared to the PBS controls. Mortality increased at day 3 but was still significantly different from the PBS controls. Mx1 transcription was significantly higher only at day 1. In an additional study Mx1 transcription was monitored in the liver, kidney, gills, spleen, and intestine at various time points post-stimulation with either poly I:C or CCV. Mx1 mRNA was significantly elevated in all organs only at day 1 post-injection with poly I:C. In response to CCV, Mx1 transcription was not significantly elevated until day 3 post-challenge, but remained elevated in certain organs until day 7.

Draft Genome Sequences of Streptococcus bovis Strains ATCC 33317 and JB1.

We report the draft genome sequences of Streptococcus bovis strain ATCC 33317 (CVM42251) isolated from cow dung and strain JB1 (CVM42252) isolated from a cow rumen in 1977. The strains were sequenced using the Genome Sequencer FLX 454 system. The genome sizes are approximately 2 Mb and 2.2 Mb, respectively.

Antimicrobial resistance genes in multidrug-resistant Salmonella enterica isolated from animals, retail meats, and humans in the United States and Canada.

Salmonella enterica is a prevalent foodborne pathogen that can carry multidrug resistance (MDR) and pose a threat to human health. Identifying the genetics associated with MDR in Salmonella isolated from animals, foods, and humans can help determine sources of MDR in food animals and their impact on humans. S. enterica serovars most frequently carrying MDR from healthy animals, retail meats, and human infections in the United States and Canada were identified and isolates resistant to the largest number of antimicrobials were chosen. Isolates were from U.S. slaughter (n=12), retail (9), and humans (9), and Canadian slaughter (9), retail (9), and humans (8; total n=56). These isolates were assayed by microarray for antimicrobial resistance and MDR plasmid genes. Genes detected encoded resistance to aminoglycosides (alleles of aac, aad, aph, strA/B); beta-lactams (bla(TEM), bla(CMY), bla(PSE-1)); chloramphenicol (cat, flo, cmlA); sulfamethoxazole (sulI); tetracycline (tet(A, B, C, D) and tetR); and trimethoprim (dfrA). Hybridization with IncA/C plasmid gene probes indicated that 27/56 isolates carried one of these plasmids; however, they differed in several variable regions. Cluster analysis based on genes detected separated most of the isolates into two groups, one with IncA/C plasmids and one without IncA/C plasmids. Other plasmid replicons were detected in all but one isolate, and included I1 (25/56), N (23/56), and FIB (10/56). The presence of different mobile elements along with similar resistance genes suggest that these genetic elements may acquire similar resistance cassettes, and serve as multiple sources for MDR in Salmonella from food animals, retail meats, and human infections.

Development and validation of a resistance and virulence gene microarray targeting Escherichia coli and Salmonella enterica.

A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing inter-laboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-operator characteristic curve=0.97). Inter-laboratory agreement, however, was poor when estimated using the intraclass correlation coefficient, which ranged from 0.27 (95% confidence interval 0.24, 0.29) to 0.29 (0.23, 0.34). These findings suggest that extensive testing and procedure standardization will be needed before bacterial genotyping arrays can be readily shared between laboratories.