Analysis of Salmonella enterica serovar Typhimurium variable-number tandem-repeat data for public health investigation based on measured mutation rates and whole-genome sequence comparisons.

Variable-number tandem repeats (VNTRs) mutate rapidly and can be useful markers for genotyping. While multilocus VNTR analysis (MLVA) is increasingly used in the detection and investigation of food-borne outbreaks caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) and other bacterial pathogens, MLVA data analysis usually relies on simple clustering approaches that may lead to incorrect interpretations. Here, we estimated the rates of copy number change at each of the five loci commonly used for S. Typhimurium MLVA, during in vitro and in vivo passage. We found that loci STTR5, STTR6, and STTR10 changed during passage but STTR3 and STTR9 did not. Relative rates of change were consistent across in vitro and in vivo growth and could be accurately estimated from diversity measures of natural variation observed during large outbreaks. Using a set of 203 isolates from a series of linked outbreaks and whole-genome sequencing of 12 representative isolates, we assessed the accuracy and utility of several alternative methods for analyzing and interpreting S. Typhimurium MLVA data. We show that eBURST analysis was accurate and informative. For construction of MLVA-based trees, a novel distance metric, based on the geometric model of VNTR evolution coupled with locus-specific weights, performed better than the commonly used simple or categorical distance metrics. The data suggest that, for the purpose of identifying potential transmission clusters for further investigation, isolates whose profiles differ at one of the rapidly changing STTR5, STTR6, and STTR10 loci should be collapsed into the same cluster.

Emergence of a ribotype 244 strain of Clostridium difficile associated with severe disease and related to the epidemic ribotype 027 strain.

BACKGROUND: We identified 12 patients with Clostridium difficile infection between July 2011 and March 2012 from whom an unusual C. difficile strain was isolated. This strain had a single-nucleotide deletion of the tcdC gene at position 117 and binary toxin genes, which are characteristic of the hypervirulent ribotype (RT) 027 strain. METHODS: A retrospective cohort study of 12 patients infected with C. difficile RT244 and 24 patients infected with non-RT244/non-RT027 strains matched for place of diagnosis and time of collection of specimen was performed. We performed whole-genome sequencing to understand the relationship of the RT244 strain to other C. difficile strains and further understand its virulence potential. RESULTS: Clostridium difficile RT244 was associated with more severe disease and a higher mortality rate. Phylogenomic analysis using core genome single-nucleotide polymorphisms showed that RT244 is in the same genetic clade (clade 2) as RT027 but is distinct from all RT027 strains. The pathogenicity locus of the RT244 strain encodes a variant toxin B, and this was confirmed by demonstration of Clostridium sordellii-like cytopathic effect on Vero cells. Toxin B production in culture supernatants was lower than that seen with a RT027 strain. CONCLUSIONS: Our findings demonstrate the pathogenic potential of this RT244 C. difficile strain and emphasize the importance of ongoing surveillance for emergent strains.

Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia.

Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008-2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (Prn). Multiple mechanisms of Prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of Prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing Prn arose independently multiple times since 2008, rather than by expansion of a single Prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.

First genetic analysis of Cryptosporidium from humans from Tasmania, and identification of a new genotype from a traveller to Bali.

Little is known about the molecular composition of Cryptosporidium species from humans living in the insular state of Tasmania, Australia. In the present study, we genetically characterized 82 samples of Cryptosporidium from humans following conventional coproscopic testing in a routine, diagnostic laboratory. Using a PCR-coupled single-strand conformation polymorphism (SSCP) technique, targeting portions of the small subunit rRNA (SSU), and 60 kDa glycoprotein (gp60) loci, we identified two species of Cryptosporidium, including C. hominis (subgenotypes IbA10G2, IdA16, IeA12G3T3, and IfA19G1) and C. parvum (IIaA16G1R1 and IIaA18G3), and a new operational taxonomic unit (OTU) that genetically closely resembled C. wrairi. This OTU was further characterized using markers in the actin, Cryptosporidium oocyst wall protein (COWP), and 70 kDa heat shock protein (hsp70) genes. This study provides the first characterization of species and genotypes of Cryptosporidium from Tasmania, and presents clear genetic evidence, using five independent genetic loci, for a new genotype or species of Cryptosporidium in a Tasmanian person with a recent history of travelling to Bali, Indonesia. It would be interesting to undertake detailed molecular-based studies of Cryptosporidium in Indonesia and neighbouring countries, in conjunction with morphological and experimental investigations of new genotypes.

Double-locus sequence typing using porA and peb1A for epidemiological studies of Campylobacter jejuni.

Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis worldwide. Bacterial typing schemes play an important role in epidemiological investigations to trace the source and route of transmission of the infectious agent by identifying outbreak and differentiating among sporadic infections. In this study, a double-locus sequence typing (DLST) scheme for C. jejuni based on concatenated partial sequences of porA and peb1A genes is proposed. The DLST scheme was validated using 50 clinical and environmental C. jejuni strains isolated from human (C5, H, H15-H19), chicken (CH1-CH15), water (W2-W17), and ovine samples (OV1-OV6). The scheme was found to be highly discriminatory (discrimination index [DI]=0.964) and epidemiologically concordant based on C. jejuni strains studied. The DLST showed discriminatory power above 0.95 and excellent congruence to multilocus sequence typing and can be recommended as a rapid and low-cost typing scheme for epidemiological investigation of C. jejuni. It is suggested that the DLST scheme is suitable for identification of outbreak strains and differentiation of the sporadic infection strains.

Evidence of microevolution of Salmonella Typhimurium during a series of egg-associated outbreaks linked to a single chicken farm.

BACKGROUND: The bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most frequent causes of foodborne outbreaks of gastroenteritis. Between 2005-2008 a series of S. Typhimurium outbreaks occurred in Tasmania, Australia, that were all traced to eggs originating from a single chicken farm. We sequenced the genomes of 12 isolates linked to these outbreaks, in order to investigate the microevolution of a pathogenic S. Typhimurium clone in a natural, spatiotemporally restricted population. RESULTS: The isolates, which shared a phage type similar to DT135 known locally as 135@ or 135a, formed a clade within the S. Typhimurium population with close similarity to the reference genome SL1334 (160 single nucleotide polymorphisms, or SNPs). Ten of the isolates belonged to a single clone (<23 SNPs between isolate pairs) which likely represents the population of S. Typhimurium circulating at the chicken farm; the other two were from sporadic cases and were genetically distinct from this clone. Divergence dating indicated that all 12 isolates diverged from a common ancestor in the mid 1990 s, and the clone began to diversify in 2003-2004. This clone spilled out into the human population several times between 2005-2008, during which time it continued to accumulate SNPs at a constant rate of 3-5 SNPs per year or 1x10-6 substitutions site-1 year-1, faster than the longer-term (~50 year) rates estimated previously for S. Typhimurium. Our data suggest that roughly half of non-synonymous substitutions are rapidly removed from the S. Typhimurium population, after which purifying selection is no longer important and the remaining substitutions become fixed in the population. The S. Typhimurium 135@ isolates were nearly identical to SL1344 in terms of gene content and virulence plasmids. Their phage contents were close to SL1344, except that they carried a different variant of Gifsy-1, lacked the P2 remnant found in SL1344 and carried a novel P2 phage, P2-Hawk, in place SL1344's P2 phage SopEϕ. DT135 lacks P2 prophage. Two additional plasmids were identified in the S. Typhimurium 135@ isolates, pSTM2 and pSTM7. Both plasmids were IncI1, but phylogenetic analysis of the plasmids and their bacterial hosts shows these plasmids are genetically distinct and result from independent plasmid acquisition events. CONCLUSIONS: This study provides a high-resolution insight into short-term microevolution of the important human pathogen S. Typhimurium. It indicates that purifying selection occurs rapidly in this population (≤ 6 years) and then declines, and provides an estimate for the short-term substitution rate. The latter is likely to be more relevant for foodborne outbreak investigation than previous estimates based on longer time scales.

Newly emerging clones of Bordetella pertussis carrying prn2 and ptxP3 alleles implicated in Australian pertussis epidemic in 2008-2010.

Australia is experiencing a prolonged epidemic of pertussis that began in 2008. A total of 194 Bordetella pertussis isolates collected from 2008 through 2010 were typed by single-nucleotide polymorphism (SNP) analysis, by multilocus variable number tandem repeats analysis, and by fim3, prn, and ptxP sequence analyses. Strains with 2 closely related SNP profiles carrying prn2 and ptxP3 from the recently emerged SNP cluster I predominated. The data suggest increasing selection among the B. pertussis population in Australia in favor of strains carrying prn2 and ptxP3 under the pressure of acellular vaccine-induced immunity.

Salmonellosis outbreak traced to playground sand, Australia, 2007-2009.

A community outbreak of gastroenteritis in Australia during 2007-2009 was caused by ingestion of playground sand contaminated with Salmonella enterica Paratyphi B, variant Java. The bacterium was also isolated from local wildlife. Findings support consideration of nonfood sources during salmonellosis outbreak investigations and indicate transmission through the animal-human interface.

Twenty-six years of enteric fever in Australia: an epidemiological analysis of antibiotic resistance.

OBJECTIVES: To determine incidence and trends in antibiotic resistance in Australian Salmonella enterica subspecies enterica serovars Typhi (S. Typhi) and Paratyphi (S. Paratyphi) isolates over the past 26 years. DESIGN: A retrospective analysis of consecutive microbiologically confirmed enteric fever isolates. PARTICIPANTS AND SETTING: All S. Typhi and S. Paratyphi isolates from patients diagnosed with enteric fever in Australia between 1985 and 2010. MAIN OUTCOME MEASURES: Incidence and variation in antibiotic resistance over time and according to country of origin. RESULTS: We analysed 2551 isolates, which originated from 74 countries or regions, mainly India (33%) and Indonesia (22%). The incidence among Australian residents increased from four to five before 2003 to seven cases per million person-years after 2003. Multidrug resistance (chloramphenicol, ampicillin, trimethoprim) and nalidixic acid resistance emerged rapidly from the early 1990s, with nalidixic acid resistance increasing to 70% in 2009-2010, while multidrug resistance was relatively stable at between 4% and 11%. Nalidixic acid and multidrug resistance rates are highest in isolates from the Indian subcontinent. Some countries in South-East Asia, such as Indonesia, had very low rates of resistance; however, this varied across the region. CONCLUSIONS: Nalidixic acid resistance has become widespread in enteric fever isolates from the Indian subcontinent and some parts of South-East Asia, justifying the use of ceftriaxone or azithromycin rather than ciprofloxacin as first-line treatment. However, resistance in some countries remains rare, potentially allowing treatment to be adjusted according to country of origin.

Observation of a new pattern in serogroup-related PCR typing of Listeria monocytogenes 4b isolates.

Molecular serogroup-related PCR typing has made the determination of serotypes of Listeria monocytogenes isolates easy and rapid. Amplification of selected lineage- and serotype-related genes can produce serotype patterns reflecting the four major serotypes, 1/2a, 1/2b, 1/2c, and 4b. We found that four isolates in our routine testing had a pattern with the four bands lmo0737, ORF2110, ORF2819, and prs positive, a pattern which has not been previously reported in the literature. After testing with a lineage-specific PCR, hybridization, and conventional agglutination serotyping, the isolates with the new pattern were considered to be serotype 4b.

Common ancestry and novel genetic traits of Francisella novicida-like isolates from North America and Australia as revealed by comparative genomic analyses.

Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.

Classification of Cryptosporidium species from patients with sporadic cryptosporidiosis by use of sequence-based multilocus analysis following mutation scanning.

In the present study, we analyzed genetic variation in Cryptosporidium species from humans (n = 62) with clinical cryptosporidiosis in South Australia. Sequence variation was assessed in regions within the small subunit of nuclear rRNA (p-SSU), the 70-kDa heat shock protein (p-hsp70), and the 60-kDa glycoprotein (p-gp60) genes by employing single-strand conformation polymorphism analysis and sequencing. Based on the analyses of p-SSU and p-hsp70, Cryptosporidium hominis (n = 38) and Cryptosporidium parvum (n = 24) were identified. The analysis of p-gp60 revealed eight distinct subgenotypes, classified as C. hominis IaA17R1 (n = 3), IbA9G3R2 (n = 14), IbA10G2R2 (n = 20), and IfA12G1R1 (n = 1), as well as C. parvum IIaA18G3R1 (n = 15), IIaA20G3R1 (n = 6), IIaA22G4R1 (n = 2), and IIcA5G3R2 (n = 1). Subgenotypes IaA17R1 and IIaA22G4R1 are new. Of the six other subgenotypes, IbA10G2R2, IIaA18G3R1, IIaA20G3R1, and IIcA5G3R2 were reported previously from the state of Victoria. This is the fourth record in Australia of C. parvum subgenotype IIaA18G3R1 from humans, which, to date, has been isolated only from cattle in other countries. This subgenotype might be a significant contributor to sporadic human cryptosporidiosis and may indicate a greater zoonotic contribution to the infection of humans in the area of study. Comparative analyses revealed, for the first time, the differences in the genetic makeup of Cryptosporidium populations between two relatively close, major metropolitan cities.

Salmonella Typhimurium and Salmonella Sofia: Growth in and Persistence on Eggs under Production and Retail Conditions.

Salmonellosis in Australia has been linked to eggs and egg products with specific serotypes associated with outbreaks. We compared attachment to and survival on egg shells and growth in eggs of two Salmonella serotypes, an egg outbreak associated Salmonella Typhimurium and a non-egg-associated Salmonella enterica ssp. II 1,4,12,27:b:[e,n,x] (S. Sofia). Experiments were conducted at combinations of 4, 15, 22, 37 and 42 °C. No significant differences occurred between the serotypes in maximum growth rates, which were significantly greater (P < 0.001) in egg yolk (0.427 log10 CFU/mL/h) compared to whole egg (0.312 log10 CFU/mL/h) and egg white (0.029 log10 CFU/mL/h). Attachment to egg shells varied by time (1 or 20 min) and temperature (4, 22 and 42 °C), with S. Typhimurium isolates attaching at higher levels (P < 0.05) than S. Sofia after 1 min at 4 °C and S. Typhimurium ATCC 14028 attaching at higher (P < 0.05) levels at 22 °C. Survival on egg shells was not significantly different across isolates. Salmonella serotypes behaved similarly regarding growth in egg contents, attachment to egg shells and survival on eggs, indicating that other factors more likely contributed to reasons for S. Typhimurium being implicated in multiple egg-associated outbreaks.

A platform for leveraging next generation sequencing for routine microbiology and public health use.

Even with the advent of next-generation sequencing (NGS) technologies which have revolutionised the field of bacterial genomics in recent years, a major barrier still exists to the implementation of NGS for routine microbiological use (in public health and clinical microbiology laboratories). Such routine use would make a big difference to investigations of pathogen transmission and prevention/control of (sometimes lethal) infections. The inherent complexity and high frequency of data analyses on very large sets of bacterial DNA sequence data, the ability to ensure data provenance and automatically track and log all analyses for audit purposes, the need for quick and accurate results, together with an essential user-friendly interface for regular non-technical laboratory staff, are all critical requirements for routine use in a public health setting. There are currently no systems to answer positively to all these requirements, in an integrated manner. In this paper, we describe a system for sequence analysis and interpretation that is highly automated and tackles the issues raised earlier, and that is designed for use in diagnostic laboratories by healthcare workers with no specialist bioinformatics knowledge.

Characterization of a novicida-like subspecies of Francisella tularensis isolated in Australia.

Francisella tularensis is found throughout the Northern Hemisphere, where it is associated with the disease of tularaemia in animals and humans. The isolation and identification is reported of a novicida-like subspecies of F. tularensis from a foot wound sustained in brackish water in the Northern Territory of Australia.

Immunity to poliomyelitis in Victorians.

OBJECTIVES: In view of WHO's goal for poliomyelitis eradication by 2005 and the possible introduction of IPV into the Australian Standard Vaccination Schedule, this investigation was conducted to assess current immunity to poliomyelitis across the Victorian community. METHODS: 1,775 sera collected from three population samples within the Victorian community between 1990 and 1995 were tested for neutralising antibody titres against each poliovirus type in accordance with WHO recommended procedure. RESULTS: In infants over three months and adults under 40 years, 76-100% of people in each age group were seropositive to all poliovirus types, with 90-100% seropositive to type I, 94-100% seropositive to type II and 80-97% seropositive to type III. Of the very small number of adults over 40 years tested (n = 13), 85% were seropositive to each of types I and II, and 62% to type III. 92% of vaccination histories taken and checked were confirmed, and reported immunisation rates were significantly below seropositive rates. CONCLUSIONS: According to poliovirus antibody seroprevalence, current immunity to poliomyelitis appears sufficient for herd immunity. When compared with vaccination histones, significantly more people demonstrated immunity to poliomyelitis than the number who reported having been vaccinated against it, indicating the possible role of intestinal vaccine strain poliovirus spread in maintaining high immunity levels. IMPLICATIONS: The current Australian Standard Vaccination Schedule has successfully provided protective levels of poliomyelitis immunity for the Victorian community in the absence of wild virus since at least 1972. This must be considered in assessing the future direction of Australia's poliomyelitis immunisation program.

A national quality assurance survey of Neisseria gonorrhoeae testing.

The aims of this study were to (1) conduct a national survey of Neisseria gonorrhoeae identification by National Neisseria Network (NNN) reference laboratories contributing data to the Australian Gonococcal Surveillance Programme and (2) determine the prevalence in Australia of strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays for confirmation of gonococcal nucleic acid amplification tests. Gonococcal clinical isolates referred to NNN laboratories for the first half of 2012 were screened using in-house real-time PCR assays targeting multicopy opa, porA pseudogene and cppB genes. There were 2455 clinical gonococcal isolates received in the study period; 98.6 % (2420/2455) of isolates harboured all three gene targets, 0.12 % (3/2455) were porA-negative, 0.04 % (1/2455) opa-negative and 1.14 % (28/2455) cppB-negative by PCR. Notably, no isolates were simultaneously negative for two targets. However, three isolates failed to be amplified by all three PCR methods, one isolate of which was shown to be a commensal Neisseria strain by 16S rRNA sequencing. Using PCR as the reference standard the results showed that (1) identification of N. gonorrhoeae isolates by NNN laboratories was highly specific (99.96 %) and (2) strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays are present but not widespread throughout Australia at this point in time.

Draft Genome Sequences for Ten Salmonella enterica Serovar Typhimurium Phage Type 135 Variants.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common cause of gastroenteritis in humans. Here, we report the draft genome sequences of 10 isolates of an S. Typhimurium phage type 135 variant that is linked to egg-associated outbreaks in Tasmania, Australia.

Community-acquired Klebsiella pneumoniae liver abscesses - an "emerging disease" in Australia.

Liver abscess due to Klebsiella pneumoniae infection has been widely reported in Asia, but rarely reported in Australia until now. We describe four previously well Asian-born patients who presented across Australia with community-acquired K. pneumoniae liver abscesses. With prompt recognition, appropriate antibiotics and early drainage, outcome is significantly improved, although vigilance for metastatic complications is essential.