A designed experiments approach to optimizing MALDI-TOF MS spectrum processing parameters enhances detection of antibiotic resistance in Campylobacter jejuni

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.IS

Multilocus sequence typing methods for the emerging Campylobacter species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus

Multilocus sequence typing (MLST) systems have been reported previously for multiple food - and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.IS

Common genomic features of Campylobacter jejuni subsp. doylei strains distinguish them from C. jejuni subsp. jejuni

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>has been divided into two subspecies: <it>C. jejuni </it>subsp. <it>jejuni </it>(<it>Cjj</it>) and <it>C. jejuni </it>subsp. <it>doylei </it>(<it>Cjd</it>). Nearly all of the <it>C. jejuni </it>strains isolated are <it>Cjj</it>; nevertheless, although <it>Cjd </it>strains are isolated infrequently, they differ from <it>Cjj </it>in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of <it>Cjd </it>strains.</p> <p>Results</p> <p>A geographically diverse collection of eight <it>Cjd </it>strains was examined by MLST and determined to be phylogenetically distinct from <it>Cjj </it>strains. Microarray-based CGI approach also supported this. We were able to demonstrate that <it>Cjd </it>strains exhibited divergence from <it>Cjj </it>strains NCTC 11168 and RM1221 in many of the intraspecies hypervariable regions. Moreover, multiple metabolic, transport and virulence functions (e.g. cytolethal distending toxin) were shown to be absent in the <it>Cjd </it>strains examined.</p> <p>Conclusion</p> <p>Our data demonstrate that <it>Cjd </it>are phylogenetically distinct from <it>Cjj </it>strains. Using the CGI approach, we identified subsets of absent genes from amongst the <it>C. jejuni </it>genes that provide clues as to the potential evolutionary origin and unusual pathogenicity of <it>Cjd</it>.</p

Characterisation of campylobacter concisus strains from South Africa using amplified fragment length polymorphism (AFLP) profiling and a genomospecies-specific polymerase chain reaction (PCR) assay: Identification of novel genomospecies and correlation with clinical data

Amplified Fragment Length Polymorphism (AFLP) profiling was used to evaluate the distribution of phenotypically indistinguishable, but genetically distinct, among Campylobacter concisus strains from South Africa. A Polymerase Chain Reaction (PCR) assay described for identifying strains belonging to Genomospecies 1 and 2 was applied in this study. Forty-seven C. concisus strains were studied in total, of which 42 were of South African origin. Forty of the South African isolates were assigned to the major existing genomospecies typified by the type strain of oral origin (GS1), and reference strains from bloody diarrhoea (GS2). Eighteen South African isolates were distributed in the GS1 cluster including two oral strains. Twenty-two faecal South African isolates clustered with reference GS2 strains. Two novel genomospecies (GS 5 and 6) were inferred by their AFLP profile characteristics. Use of an existing PCR assay first described for identification of GS1 and GS2 strains generally indicated that the tool was accurate, although the novel genomospecies described here yielded an amplicon in the GS2 assay. No consistent clinical pattern among the diarrhoea South African strains could be discerned. The study extends the known genetic diversity among C. concisus, elucidates the presence of multiple genomospecies in South Africa, and confirms for the first time an association of GS1 with diarrhoea as well as the utility (with caveats) of a PCR assay for identifying GS1 and GS2 strains

Multilocus Sequence Typing Methods for the Emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus

Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species

Comparative Genomic Analysis of Clinical Strains of Campylobacter jejuni from South Africa

BACKGROUND: Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the post-infectious neuropathies, Guillain-Barré and Miller Fisher syndromes. In the Cape Town area of South Africa, C. jejuni strains with Penner heat-stable (HS) serotype HS:41 have been observed to be overrepresented among cases of Guillain-Barré syndrome. The present study examined the genetic content of a collection of 32 South African C. jejuni strains with different serotypes, including 13 HS:41 strains, that were recovered from patients with enteritis, Guillain-Barré or Miller Fisher syndromes. The sequence-based typing methods, multilocus sequence typing and DNA microarrays, were employed to potentially identify distinguishing features within the genomes of these C. jejuni strains with various disease outcomes. METHODOLOGY/PRINCIPAL FINDINGS: Comparative genomic analyses demonstrated that the HS:41 South African strains were clearly distinct from the other South African strains. Further DNA microarray analysis demonstrated that the HS:41 strains from South African patients with the Guillain-Barré syndrome or enteritis were highly similar in gene content. Interestingly, the South African HS:41 strains were distinct in gene content when compared to HS:41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements (CJIEs). Only the integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS:41 strains whereas this element was absent in two closely-related HS:41 strains from Mexico. A more distantly-related HS:41 strain from Canada possessed both integrated elements CJIE1 and CJIE2. CONCLUSION/SIGNIFICANCE: These findings demonstrate that CJIEs may contribute to the differentiation of closely-related C. jejuni strains. In addition, the presence of bacteriophage-related genes in CJIE1 may contribute to the genomic diversity of C. jejuni strains. This comparative genomic analysis of C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks

doi:10.1016/j.clinmicnews.2006.03.004

Abstract Campylobacter jejuni is universally recognized as the most common bacterial cause of human gastroenteritis. This organism is also associated with septicemia, meningitis, and the post-infective sequelae of the Guillain-Barré syndrome and reactive arthritis. Interest is increasing in Campylobacter species other than C. jejuni and their roles in human and animal disease. However, these emerging campylobacteria are dramatically under-isolated because of the application of less than optimal laboratory protocols. A Cape Town protocol in which stool is filtered onto antibiotic-free culture media and incubated in a hydrogen-enriched, microaerobic atmosphere is a simple and cost-effective means of optimizing the recovery of all species of Campylobacter, as well as species of the related genera Arcobacter and Helicobacter, from stool, blood, and other clinical samples. Subsequent biochemical identification by means of a flowchart easily identifies presumptive Campylobacter isolates to the species level. As emerging Campylobacter spp. are dramatically under-isolated, the true disease potential of these organisms is unknown at present. , and C. curvus, have an essential growth requirement for hydrogen or formate These Campylobacter species have been isolated from the stools of patients suffering from gastroenteritis, septicemia, and other clinical conditions (3). Hydrogen-dependent organisms are extremely difficult, if not impossible, to recover by the standard culture techniques currently employed in most routine diagnostic laboratories. Some diagnostic laboratories use 42°C as a primary incubation temperature, which allows growth of C. jejuni subsp. jejuni and C. coli but not of other species, such as C. fetus or C. hyointestinalis, which grow at 37°C but not 42°C. C. jejuni subsp. jejuni and C. coli contaminate chicken which has an internal temperature of 42°C. This is probably the historical precedent for using 42°C as a primary incubation temperature. For clinical specimens, it is recommended that incubation at 37°C is a more appropriate temperature, as all Campylobacter spp. infecting humans can be isolated and maintained at this temperature. Up to 17% of clinical samples may have two to five distinct species of Campylobacter or species of the related genera Arcobacter and Helicobacter identified on the primary isolation plate (4). C. jejuni subsp. jejuni may be coisolated with an emerging Campylobacter sp. Different species of Campylobacter and related genera can often be detected initially by differences in colony morpholog