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.

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Molecular Typing of Salmonella enterica Serovar Typhi Isolates from Various Countries in Asia by a Multiplex PCR Assay on Variable-Number Tandem Repeats

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.

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

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA was performed on 133 strains of Salmonella enterica serovar Typhi obtained from Papua New Guinea, with the objective of assessing the temporal variation of these strains. Fifty-two strains that were isolated in 1992 and 1994 were of one phage type, D2, and only two predominant PFGE profiles, X1 and X2, were present. Another 81 strains isolated between 1997 and 1999 have shown divergence, with four new phage types, UVS I (n = 63), UVS (n = 5), VNS (n = 4), and D1 (n = 9), and more genetic variability as evidenced by the multiple and new PFGE XbaI profiles (21 profiles; Dice coefficient, F = 0.71 to 0.97). The two profiles X1 and X2 have remained the stable, dominant subtypes since 1992. Cluster analysis based on the unweighted pair group method using arithmetic averages algorithm identifies two main clusters (at 87% similarity), indicating that the divergence of the PFGE subtypes was probably derived from some genomic mutations of the X1 and X2 subtypes. The majority of isolates were from patients with mild and moderate typhoid fever and had various XbaI profiles. A single isolate from a patient with fatal typhoid fever had a unique X11 profile, while four of six isolates from patients with severe typhoid fever had the X1 pattern. In addition, 12 paired serovar Typhi isolates recovered from the blood and fecal swabs of individual patients exhibited similar PFGE patterns, while in another 11 individuals paired isolates exhibited different PFGE patterns. Three pairs of isolates recovered from three individuals had different phage types and PFGE patterns, indicating infection with multiple strains. The study reiterates the usefulness of PFGE in assessing the genetic diversity of S. enterica serovar Typhi for both long-term epidemiology and in vivo stability and instability within an individual patient.

Increasing genetic diversity of Salmonella enterica serovar Typhi isolates from Papua New Guinea over the period from 1992 to 1999

Y . L . G O H , R . Y A S I N , S . D . P U T H U C H E A R Y , Y . T . K O H , V . K . E . L I M , Z . T A I B A N D K . L . T H O N G . 2003. Aims: DNA fingerprinting of Salmonella enterica serotype Paratyphi B isolated in Malaysia during 1982‐83, 1992 and 1996‐2002 was carried out by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility tests and D-tartrate utilization tests to assess the extent of genetic diversity of these isolates in Malaysia. Methods and Results: Eighty-six human isolates and one food isolate of Salm. Paratyphi B were analysed by PFGE, antimicrobial susceptibility tests and D-tartrate utilization tests. Sixty-five strains were d-tartratenegative (dT)) while 22 strains were D-tartrate-positive (dT+). Thirty-seven per cent of the Salm. Paratyphi B strains were resistant to one or more antimicrobial agents. PFGE analysis clearly distinguished the dT) and dT+ strains into two clusters based on the unweighted pair group average method (UPGMA). Twenty-two XbaI-pulsotypes were observed among the 65 dT) strains while 17 XbaI-pulsotypes were observed among the 22 isolates of Salm. Paratyphi B dT+. Conclusions: The present study showed that PFGE was very discriminative with 33AE7% of the strains yielding distinct fingerprints. Paratyphoid fever in Malaysia is probably caused by one predominant, endemic clone of Salm. Paratyphi B dT) with various subtypes. There was no association between the pulsotypes and the severity of the disease indicating that the severity of the disease is probably multifactorial. Significance and Impact of the Study: The findings of the present study verify the usefulness of PFGE in characterizing strains of Salm. Paratyphi B. This is the first report on the application of PFGE on a large collection of Salm. Paratyphi B in Malaysia.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2672.2003.02107.x

Yee-Ling Goh

Papers

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

Increasing genetic diversity of Salmonella enterica serovar Typhi isolates from Papua New Guinea over the period from 1992 to 1999

DNA fingerprinting of human isolates of Salmonella enterica serotype Paratyphi B in Malaysia