Nosocomial infections pose a significant threat to patient health; however, the gold standard laboratory method for determining bacterial relatedness (pulsed-field gel electrophoresis [PFGE]) remains essentially unchanged 20 years after its introduction. Here, we explored bacterial whole-genome sequencing (WGS) as an alternative approach for molecular strain typing. We compared WGS to PFGE for investigating presumptive outbreaks involving three important pathogens: vancomycin-resistant Enterococcus faecium (n = 19), methicillin-resistant Staphylococcus aureus (n = 17), and Acinetobacter baumannii (n = 15). WGS was highly reproducible (average ≤ 0.39 differences between technical replicates), which enabled a functional, quantitative definition for determining clonality. Strain relatedness data determined by PFGE and WGS roughly correlated, but the resolution of WGS was superior (P = 5.6 × 10−8 to 0.016). Several discordant results were noted between the methods. A total of 28.9% of isolates which were indistinguishable by PFGE were nonclonal by WGS. For A. baumannii, a species known to undergo rapid horizontal gene transfer, 16.2% of isolate pairs considered nonidentical by PFGE were clonal by WGS. Sequencing whole bacterial genomes with single-nucleotide resolution demonstrates that PFGE is prone to false-positive and false-negative results and suggests the need for a new gold standard approach for molecular epidemiological strain typing.

Application of Whole-Genome Sequencing for Bacterial Strain Typing in Molecular Epidemiology

We serotyped 44,087 Salmonella isolates from humans and 26,148 from other sources from 1993 through 2002. The most common serovar causing human salmonellosis in Thailand was Salmonella enterica Weltevreden. Serovars causing human infections in Thailand differ from those in other countries and seem to be related to Salmonella serovars in different food products and reservoirs.

Salmonella serovars from humans and other sources in Thailand, 1993-2002.

Objectives: This study was conducted to investigate the occurrence of antimicrobial resistance among Sal- monella Weltevreden isolates from different sources in South-East Asia (Indonesia, Laos, Malaysia, Taiwan, Thailand, Vietnam), Australia, Denmark, New Zealand and the USA. Methods: A total of 503 isolates were examined for susceptibility to antimicrobial agents, and resistant isolates were examined for the presence of selected resistance genes by PCR. Results: Only 48 (9.5%) of the isolates were resistant to one or more of the antimicrobial agents tested. A low frequency of resistance was found towards ampicillin (1.8%), chloramphenicol (1.6%), florphenicol (0.4%), nalidixic acid (1.6%), neomycin (0.6%), streptomycin (4.4%), sulfamethoxazole (4.2%), tetracycline (4.0%) and trimethoprim (1.4%), whereas all isolates were susceptible to co-amoxiclav, ceftiofur, ciprofloxacin, col- istin and gentamicin. All nine ampicillin-resistant isolates contained a sequence similar to the blaTEM-1b gene, one of the eight chloramphenicol-resistant isolates a sequence similar to the catA1 gene, all three neomycin- resistant isolates a sequence similar to the aphA-2 gene, 16 (73%) of the 22 streptomycin-resistant isolates a sequence similar to the aadA gene, the remaining six (27%) a sequence similar to the strA gene, and all 21 sulfamethoxazole-resistant isolates a sequence similar to the sul2 gene. Thirteen (65%) of the 20 tetracycline- resistant isolates contained the tet(A) gene, four (20%) the tet(B) gene, and one (5%) the tet(C) gene. Conclusions: This study showed a low frequency of resistance among Salmonella Weltevreden isolated from humans and other reservoirs in South-East Asia and elsewhere. There was no major difference in the occurrence of resistance between source or geographical origin.

Antimicrobial susceptibility and occurrence of resistance genes among Salmonella enterica serovar Weltevreden from different countries

Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood.

Non-typhoidal Salmonella is the most common foodborne bacterial pathogen in most countries. It is widely present in food animal species, and therefore blocking its transmission through the food supply is a prominent focus of food safety activities worldwide. Antibiotic resistance in non-typhoidal Salmonella arises in large part because of antibiotic use in animal husbandry. Tracking resistance in Salmonella is required to design targeted interventions to contain or diminish resistance and refine use practices in production. Many countries have established systems to monitor antibiotic resistance in Salmonella and other bacteria, the earliest ones appearing the Europe and the US. In this chapter, we compare recent Salmonella antibiotic susceptibility data from Europe and the US. In addition, we summarize the state of known resistance genes that have been identified in the genus. The advent of routine whole genome sequencing has made it possible to conduct genomic surveillance of resistance based on DNA sequences alone. This points to a new model of surveillance in the future that will provide more definitive information on the sources of resistant Salmonella, the specific types of resistance genes involved, and information on how resistance spreads.

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Antimicrobial Resistance in Nontyphoidal Salmonella.

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.

Y.T. Koh

Papers

Genetic diversity of clinical and environmental strains of Salmonella enterica serotype Weltevreden isolated in Malaysia.