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Escherichia coli sequence type 95 (ST95) is an extraintestinal pathogenic E. coli (ExPEC) renowned for its ability to cause significant morbidity and mortality in humans and poultry. A core genome analysis of 668 ST95 isolates generated 10 clades (A to J), 5 of which are reported here for the first time. F plasmid replicon sequence typing showed that almost a third (178/668 [27%]) of the collection carry pUTI89 (F29:B10) and were restricted to clade A and a sublineage of clade B. In contrast, almost half (328/668 [49%]) of the collection across multiple clades harbor ColV plasmids (multiple F types). Strikingly, ST95 lineages with pUTI89 were almost exclusively from humans, while ColV+ ST95 lineages were sourced from poultry and humans. Clade I was notable because it comprises temporally and geographically matched ColV+ isolates sourced from human and retail poultry meat, suggesting interspecies transmission via food. Clade F contained ST95 isolates of bovine origin, none of which carried ColV or pUTI89 plasmids. Remarkably, an analysis of a cohort of 34,176 E. coli isolates comprising 2,570 sequence types mirrored what was observed in ST95: (i) pUTI89 was overwhelmingly linked to E. coli sourced from humans but almost entirely absent from 13,027 E. coli isolates recovered from poultry, pigs, and cattle, and (ii) E. coli isolates harboring ColV plasmids were from multiple sources, including humans, poultry, and swine. Overall, our data suggest that F plasmids influence E. coli host range, clade structure, and zoonotic potential in ST95 and ExPEC more broadly. IMPORTANCE E. coli ST95 is one of five dominant ExPEC lineages globally and noted for causing urinary tract and bloodstream infections and neonatal meningitis in humans and colibacillosis in poultry. Using high-resolution phylogenomics, we show that F replicon sequence type is linked to ST95 clade structure and zoonotic potential. Specifically, human centric ST95 clades overwhelmingly harbor F29:B10 (pUTI89) plasmids, while clades carrying both human- and poultry-sourced isolates are typically ColV+ with multiple replicon types. Importantly, several clades identified clonal ColV+ ST95 isolates from human and poultry sources, but clade I, which housed temporally and spatially matched isolates, provided the most robust evidence. Notably, patterns of association of F replicon types with E. coli host were mirrored within a diverse collection of 34,176 E. coli genomes. Our studies indicate that the role of food animals as a source of human ExPEC disease is complex and warrants further investigation.

Using a previously described metagenomics dataset of 27 billion reads, we reconstructed over 50 000 metagenome-assembled genomes (MAGs) of organisms resident in the porcine gut, 46.5 % of which were classified as >70 % complete with a

Escherichia coli ST216, including those that carry blaKPC-2, blaFOX-5, blaCTX-M-15 and mcr-1, have been linked to wild and urban-adapted birds and the colonisation of hospital environments causing recalcitrant, carbapenem-resistant human infections. Here we sequenced 22 multiple-drug resistant ST216 isolates from Australian silver gull chicks sampled from Five Islands, of which 21 carried nine or more antibiotic resistance genes including blaIMP-4 (n = 21), blaTEM-1b (n = 21), aac(3)-IId (n = 20), mph(A) (n = 20), catB3 (n = 20), sul1 (n = 20), aph(3")-Ib (n = 18) and aph(6)-Id (n = 18) on FIB(K) (n = 20), HI2-ST1 (n = 11) and HI2-ST3 (n = 10) plasmids. We show that (i) all HI2 plasmids harbour blaIMP-4 in resistance regions containing In809 flanked by IS26 (HI2-ST1) or IS15DI (HI2-ST3) and diverse metal resistance genes; (ii) HI2-ST1 plasmids are highly related to plasmids reported in diverse Enterobacteriaceae sourced from humans, companion animals and wildlife; (iii) HI2 were a feature of the Australian gull isolates and were not observed in international ST216 isolates. Phylogenetic analyses identified close relationships between ST216 from Australian gull and clinical isolates from overseas. E. coli ST216 from Australian gulls harbour HI2 plasmids encoding resistance to clinically important antibiotics and metals. Our studies underscore the importance of adopting a one health approach to AMR and pathogen surveillance.

Although most of the approximately 94 million annual human cases of gastroenteritis due to Salmonella enterica resolve without medical intervention, antimicrobial therapy is recommended for patients with severe disease. Wild birds can be natural hosts of Salmonella that pose a threat to human health; however, multiple-drug-resistant serovars of S. enterica have rarely been described. In 2012, silver gull (Chroicocephalus novaehollandiae) chicks at a major breeding colony were shown to host Salmonella, most isolates of which were susceptible to antibiotics. However, multiple-drug-resistant (MDR) Escherichia coli with resistance to carbapenems, ceftazidime, and fluoroquinolones was reported from this breeding colony. In this paper, we describe a novel MDR Salmonella strain subsequently isolated from the same breeding colony. SG17-135, an isolate of S. enterica with phenotypic resistance to 12 individual antibiotics but only nine antibiotic classes including penicillins, cephalosporins, monobactams, macrolides, fluoroquinolones, aminoglycosides, dihydrofolate reductase inhibitors (trimethoprim), sulfonamides, and glycylcyclines was recovered from a gull chick in 2017. Whole-genome sequence (WGS) analysis of SG17-135 identified it as Salmonella enterica serovar Agona (S Agona) with a chromosome comprising 4,813,284 bp, an IncHI2 ST2 plasmid (pSG17-135-HI2) of 311,615 bp, and an IncX1 plasmid (pSG17-135-X) of 27,511 bp. pSG17-135-HI2 housed a complex resistance region comprising 16 antimicrobial resistance genes including blaCTX-M-55 The acquisition of MDR plasmids by S. enterica described here poses a serious threat to human health. Our study highlights the importance of taking a One Health approach to identify environmental reservoirs of drug-resistant pathogens and MDR plasmids.IMPORTANCE Defining environmental reservoirs hosting mobile genetic elements that shuttle critically important antibiotic resistance genes is key to understanding antimicrobial resistance (AMR) from a One Health perspective. Gulls frequent public amenities, parklands, and sewage and other waste disposal sites and carry drug-resistant Escherichia coli Here, we report on SG17-135, a strain of Salmonella enterica serovar Agona isolated from the cloaca of a silver gull chick nesting on an island in geographic proximity to the greater metropolitan area of Sydney, Australia. SG17-135 is closely related to pathogenic strains of S Agona, displays resistance to nine antimicrobial classes, and carries important virulence gene cargo. Most of the antibiotic resistance genes hosted by SG17-135 are clustered on a large IncHI2 plasmid and are flanked by copies of IS26 Wild birds represent an important link in the evolution and transmission of resistance plasmids, and an understanding of their behavior is needed to expose the interplay between clinical and environmental microbial communities.

Urinary tract infections (UTIs) are the most common bacterial infections requiring medical attention and a leading justification for antibiotic prescription. Trimethoprim is prescribed empirically for uncomplicated cases. UTIs are primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) and ExPEC strains play a central role in disseminating antimicrobial-resistance genes worldwide. Here, we describe the whole-genome sequences of trimethoprim-resistant ExPEC and/or ExPEC from recurrent UTIs (67 in total) from patients attending a regional Australian hospital from 2006 to 2008. Twenty-three sequence types (STs) were observed, with ST131 predominating (28 %), then ST69 and ST73 (both 7 %). Co-occurrence of trimethoprim-resistance genes with genes conferring resistance to extended-spectrum β-lactams, heavy metals and quaternary ammonium ions was a feature of the ExPEC described here. Seven trimethoprim-resistance genes were identified, most commonly dfrA17 (38 %) and dfrA12 (18 %). An uncommon dfrB4 variant was also observed. Two blaCTX-M variants were identified - blaCTX-M-15 (16 %) and blaCTX-M-14 (10 %). The former was always associated with dfrA12, the latter with dfrA17, and all blaCTX-M genes co-occurred with chromate-resistance gene chrA. Eighteen class 1 integron structures were characterized, and chrA featured in eight structures; dfrA genes featured in seventeen. ST131 H30Rx isolates possessed distinct antimicrobial gene profiles comprising aac(3)-IIa, aac(6)-Ib-cr, aph(3')-Ia, aadA2, blaCTX-M-15, blaOXA-1 and dfrA12. The most common virulence-associated genes (VAGs) were fimH, fyuA, irp2 and sitA (all 91 %). Virulence profile clustering showed ST131 H30 isolates carried similar VAGs to ST73, ST405, ST550 and ST1193 isolates. The sole ST131 H27 isolate carried molecular predictors of enteroaggregative E. coli/ExPEC hybrid strains (aatA, aggR, fyuA). Seven isolates (10 %) carried VAGs suggesting ColV plasmid carriage. Finally, SNP analysis of serial UTI patients experiencing worsening sequelae demonstrated a high proportion of point mutations in virulence factors.

Silver gulls carry phylogenetically diverse Escherichia coli including globally dominant ExPEC sequence types and pandemic ExPEC-ST131 clades, however our large-scale study (504 samples) on silver gulls nesting off the coast of New South Wales identified E. coli ST457 as the most prevalent. A phylogenetic analysis of whole-genome sequences (WGS) of 138 ST457 comprising of 42 from gulls, two from humans (Australia) and 14 from poultry farmed in Paraguay were compared with 80 WGS deposited in public databases from diverse sources and countries. E. coli ST457 strains are phylogenetic group F, carry fimH145 and partition into five main clades in accordance to predominant flagella H-antigen carriage. Although we identified considerable phylogenetic diversity among the 138 ST457 strains, closely related subclades (< 100 SNPs) suggested zoonotic or zooanthroponosis transmission between humans, wild birds and food-producing animals. Australian human clinical and gull strains in two of the clades were closely related (≤ 80 SNPs). Regarding plasmid content, country or country-source specific connections were observed including I1/ST23, I1/ST314 and I1/ST315 disseminating bla CMY-2 in Australia, I1/ST113 carrying bla CTX-M-8 and mcr-5 in Paraguayan poultry and F2:A-:B1 plasmids of Dutch origin across multiple ST457 clades. We identified a high prevalence of nearly identical I1/ST23 plasmids carrying bla CMY-2 among Australian gull and clinical human strains. In summary, ST457 is a broad host range, geographically-diverse E. coli lineage that can cause human extraintestinal disease including urinary tract infection and displays a remarkable ability to capture mobile elements that carry and transmit genes encoding resistance to critically important antibiotics.

The H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed extra-pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum ß-lactams. Here we describe isolates EC233 and EC234, both variants of ST131-H30Rx with a novel sequence type (ST) 8196, from unrelated patients presenting with bacteraemia at Concord Repatriation Hospital in Sydney in 2014. EC233 and EC234 are phylogroup B2, serotype O25:H4A, resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both isolates carry an IncFII_2 plasmid similar to pSPRC_Ec234-FII (85,199 bp characterised in EC234), two small plasmids and a novel IncI1 plasmid similar to pSPRC_Ec234-I (92,955 bp characterised in EC234). Apart from a chromosomally located bla CTX-M-15 module, the resistance genes are flanked by IS26 and form a complex resistance locus (CRL) on pSPRC_Ec234-FII. SNP-based phylogenetic analysis of the core genome of all ST representatives within the ST131 clonal complex places both isolates in a small subclade with 3 other clinical Australian ST131-H30Rx clade C isolates. MrBayes phylogeny analysis of ST8196 using a global collection of ST131 genomes indicated EC233 and EC234 share a most recent common ancestor with EC70, a MDR ST131-H30Rx clone, isolated from the same Sydney hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in both the ST8196 isolates and highlights the requirement for unbiased genomic surveillance approaches to identify and track novel high-risk MDR E. coli pathogens that impact healthcare facilities.

Antibiotic resistance genes (ARGs) including those from the blaCTX-M family and mcr-1 that encode resistance to extended spectrum β-lactams and colistin, respectively, have been linked with IncHI2 plasmids isolated from swine production facilities globally but not in IncHI2 plasmids from Australia. Here we describe the first complete sequence of a multiple drug resistance Australian IncHI2-ST4 plasmid, pTZ41_1P, from a commensal E. coli from a healthy piglet. pTZ41_1P carries genes conferring resistance to heavy-metals (copper, silver, tellurium and arsenic), β-lactams, aminoglycosides and sulphonamides. The ARGs reside within a complex resistance locus (CRL) that shows considerable sequence identity to a CRL in pSDE_SvHI2, an IncHI2:ST3 plasmid from an enterotoxigenic E. coli with serotype O157:H19 of porcine origin that caused substantial losses to swine production operations in Australia in 2007. pTZ41_1P is closely related to IncHI2 plasmids found in E. coli and Salmonella enterica from porcine, avian and human sources in Europe and China but it does not carry genes encoding resistance to clinically-important antibiotics. We identified regions of IncHI2 plasmids that contribute to the genetic plasticity of this group of plasmids and highlight how they may readily acquire new resistance gene cargo. Genomic surveillance should be improved to monitor IncHI2 plasmids.

Wildlife, and birds in particular, play an increasingly recognized role in the evolution and transmission of Escherichia coli that pose a threat to humans. To characterize these lineages and their potential threat from an evolutionary perspective, we isolated and performed whole-genome sequencing on 11 sequence types (STs) of E. coli recovered from the desiccated faeces of straw-necked ibis (Threskiornis spinicollis) nesting on inland wetlands located in geographically different regions of New South Wales, Australia. Carriage of virulence-associated genes was limited, and no antimicrobial resistance genes were detected, but novel variants of an insertion element that plays an important role in capturing and mobilizing antibiotic resistance genes, IS26, were identified and characterized. The isolates belonged to phylogroups B1 and D, including types known to cause disease in humans and animals. Specifically, we found E. coli ST58, ST69, ST162, ST212, ST446, ST906, ST2520, ST6096 and ST6241, and a novel phylogroup D strain, ST10208. Notably, the ST58 strain hosted significant virulence gene carriage. The sequences of two plasmids hosting putative virulence-associated factors with incompatibility groups I1 and Y, an extrachromosomal integrative/conjugative element, and a variant of a large Escherichia phage of the family Myoviridae, were additionally characterized. We identified multiple epidemiologically relevant gene signatures that link the ibis isolates to sequences from international sources, plus novel variants of IS26 across different sequence types and in different contexts.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis resulting in economic losses in the poultry industry worldwide. A total of 168 APEC isolates, equal numbers from Australian and Thai broilers/broiler breeders, were identified and tested for their susceptibility to ten antimicrobial agents. Most of the Thai APEC isolates were multidrug-resistant (MDR) (60.7%) whilst Australian APEC isolates showed a MDR rate of just 10.7%. The Thai APEC isolates exhibited high resistance to tetracycline (TET) (84.5%), amoxicillin (AMX) (70.2%) and trimethoprim-sulfamethoxazole (SXT) (51.2%) whilst the Australian APEC isolates showed lower levels of resistance (TET 36.9%, AMX 29.8%, SXT 17.86%). The 34 Thai APEC and four Australian APEC isolates which were resistant to nalidixic acid were characterized for their carriage of mutations in the quinolone resistance determining region of gyrA, gyrB, parC and parE. While no mutations were detected in gyrB in the Thai isolates, the Ser83Leu and Asp87Asn substitutions in gyrA and Ser80Ile in parC were common (n = 9/34). In regard to the Australian isolates, the Ser83Leu and Asp678Glu substitution in gyrA, Pro385Ala and Ser492Asn in gyrB and Met241Ile and Asp475Glu in parC were identified (n = 3/4). Rep-PCR analysis of the 84 Thai and 84 Australian APEC isolates showed 16 main clusters that mostly contained isolates from both countries. Our results suggest that the emergence of MDR is a major concern for the Thai APEC isolates and that more prudent use of antimicrobial agents in Thai poultry production is required.

BACKGROUND:Infection in the oviduct (salpingitis) is the most common bacterial infection in egg laying hens and is mainly caused by Escherichia coli. The disease is responsible for decreased animal welfare, considerable economic loss as well as a risk of horizontal and vertical transmission of pathogenic E. coli. The outcome of salpingitis may be either acute or chronic. It has not yet been clarified whether the pathological manifestation is a result of the characteristics of the E. coli or whether the manifestation is associated with host factors such as host immunity. RESULTS:From the core- and accessory genome analysis and comparison of 62 E. coli no genetic markers were found to be associated to either acute or chronic infection. Twenty of the 62 genomes harboured at least one antimicrobial resistance gene with resistance against sulfonamides being the most common. The increased serum survival and iron chelating genes iss and iroN were highly prevalent in genomes from both acute and chronic salpingitis. CONCLUSION:Our analysis revealed that no genetic markers could differentiate the E. coli isolated from acute versus chronic salpingitis in egg laying hens. The difference in pathological outcome may be related to other factors such as immunological status, genetics and health of the host. These data indicate that salpingitis is another manifestation of colibacillosis.

This study sought to assess the genetic variability of Escherichia coli isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 E. coli isolates sourced from community-onset (CO) and hospital-onset (HO) BSIs. The cohort comprised 64 CO and 17 HO isolates, including 35 multidrug-resistant (MDR) isolates exhibiting phenotypic resistance to three or more antibiotic classes. Phylogenetic analysis identified two major ancestral clades. One was genetically diverse with 25 isolates distributed in 16 different sequence types (STs) representing phylogroups A, B1, B2, C and F, while the other comprised phylogroup B2 isolates in subclades representing the ST131, ST73 and ST95 lineages. Forty-seven isolates contained a class 1 integron, of which 14 carried bla CTX -M-gene. Isolates with a class 1 integron carried more antibiotic resistance genes than isolates without an integron and, in most instances, resistance genes were localized within complex resistance loci (CRL). Resistance to fluoroquinolones could be attributed to point mutations in chromosomal parC and gyrB genes and, in addition, two isolates carried a plasmid-associated qnrB4 gene. Co-resistance to fluoroquinolone and broad-spectrum beta-lactam antibiotics was associated with ST131 (HO and CO), ST38 (HO), ST393 (CO), ST2003 (CO) and ST8196 (CO and HO), a novel ST identified in this study. Notably, 10/81 (12.3 %) isolates with ST95 (5 isolates), ST131 (2 isolates), ST88 (2 isolates) and a ST540 likely carry IncFII-IncFIB plasmid replicons with a full spectrum of virulence genes consistent with the carriage of ColV-like plasmids. Our data indicate that IncF plasmids play an important role in shaping virulence and resistance gene carriage in BSI E. coli in Australia.

Extraintestinal pathogenic Escherichia coli (ExPEC) can cause urinary tract and other types of infection in cats, but the relationship of cat ExPEC to human ExPEC remains equivocal. This study investigated the prevalence of ExPEC-associated sequence types (STs) from phylogenetic group B2 among fluoroquinolone-susceptible cat clinical isolates. For this, 323 fluoroquinolone-susceptible cat clinical E. coli isolates from Australia underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 274 group B2 isolates, 53 underwent whole genome sequencing (WGS), whereas 221 underwent PCR-based screening for (group B2) sequence type complexes (STc) STc12, STc73, ST131, and STc372. Group B2 was the dominant phylogenetic group (274/323, 85 %), whereas within group B2 ST73 dominated, according to both WGS (43 % of 53; followed by ST127, ST12, and ST372 [4/53, 8 % each]) and ST-specific PCR (20 % of 221). In WGS-based comparisons of cat and reference human ST73 isolates, cat isolates had a relatively conserved virulence gene profile but were phylogenetically diverse. Although in the phylogram most cat and human ST73 isolates occupied host species-specific clusters within serotype-specific clades (O2:H1, O6:H1, O25:H1, O50/O2:H1), cat and human isolates were intermingled within two serotype-specific clades: O120:H31 (3 cat and 2 human isolates) and O22:H1 (3 cat and 5 human isolates). These findings confirm the importance of human-associated group B2 lineages as a cause of urinary tract infections in cats. The close genetic relationship of some cat and human ST73 strains suggests bi-directional transmission may be possible.

© 2020, Springer Nature Switzerland AG. Whole-genome sequencing (WGS) has significantly improved our ability to understand how, through gene acquisition, bacteria can become resistant to antibiotic therapies and cause an increasingly substantial burden of disease. In this chapter, we take the well-known indicator bacteria and opportunistic pathogen Escherichia coli, predicted to be one of the leading causes of antimicrobial resistance (AMR) infections in the next decades, and demonstrate the potential insights that can be gained using WGS and genomic epidemiology. Specifically, we discuss the mechanisms by which these bacteria acquire, retain, propagate, and disperse gene combinations with a focus on key mobile genetic elements, notably ColV/BM plasmids. Efforts are underway to further standardise and streamline WGS and resistome screening from multiple environments to support the rapidly increasing user base and facilitate regional and global public health monitoring, outbreak tracking, and AMR evolutionary prediction and preparedness. The ability of E. coli to exist in multiple environments as both a pathogen and commensal organism are central to its value for establishing meaningful One Health systems-based AMR monitoring, mitigation, and management.

© 2019 Elsevier Inc. IncHI2-ST1 plasmids play an important role in co-mobilizing genes conferring resistance to critically important antibiotics and heavy metals. Here we present the identification and analysis of IncHI2-ST1 plasmid pSPRC-Echo1, isolated from an Enterobacter hormaechei strain from a Sydney hospital, which predates other multi-drug resistant IncHI2-ST1 plasmids reported from Australia. Our time-resolved phylogeny analysis indicates pSPRC-Echo1 represents a new lineage of IncHI2-ST1 plasmids and show how their diversification relates to the era of antibiotics.

© 2019 Microbiology Australia. All rights reserved. Antimicrobial resistance (AMR) threatensmodernmedicine asweknow it.AMR infectionsmay ultimately beuntreatable and routine surgeries will become inherently risky1. By 2050 more people may die of drug-resistant infections (DRIs) every year than of cancer, which equates to more than 10 million annual deaths globally2 and the World Bank has estimated that AMR could cost the global economy 1 trillion every year after 2030.DRIs alsolead to an increase in the length of hospital stays, the use of more toxic or costly antibiotics and an increased likelihood of death3. BRIC nations (Brazil, Russia, India, China) and socio-economically challenged countries and people who already have higher rates of infectious diseases will feel the greatest impact2. Indeed, AMR has been likened to the 2008 global financial crisis on an annual repeat cycle.Thatis because the effects of AMR are not just confined to the human medical sector. The veterinary sector is also reliant on the availability of antimicrobials to treat infectious diseases in companion and food-producing animals.

© 2019 Roy Chowdhury, McKinnon, Liu and Djordjevic. Escherichia coli ST405 is an emerging urosepsis pathogen, noted for carriage of blaCTX-M, blaNDM, and a repertoire of virulence genes comparable with O25b:H4-ST131. Extraintestinal and multidrug resistant E. coli ST405 are poorly studied in Australia. Here we determined the genome sequence of a uropathogenic, multiple drug resistant E. coli ST405 (strain 2009-27) from the mid-stream urine of a hospital patient in Sydney, Australia, using a combination of Illumina and SMRT sequencing. The genome of strain 2009-27 assembled into two unitigs; a chromosome comprising 5,287,472 bp and an IncB/O plasmid, pSDJ2009-27, of 89,176 bp. In silico and phenotypic analyses showed that strain 2009-27 is a serotype O102:H6, phylogroup D ST405 resistant to ampicillin, azithromycin, kanamycin, streptomycin, trimethoprim, and sulphafurazole. The genes encoding resistance to these antibiotics reside within a novel, mobile IS26-flanked transposon, identified here as Tn6242, in the chromosomal gene yjdA. Tn6242 comprises four modules that each carries resistance genes flanked by IS26, including a class 1 integron with dfrA17 and aadA5 gene cassettes, a variant of Tn6029, and mphA. We exploited unique genetic signatures located within Tn6242 to identify strains of ST405 from Danish patients that also carry the transposon in the same chromosomal location. The acquisition of Tn6242 into yjdA in ST405 is significant because it (i) is vertically inheritable; (ii) represents a reservoir of resistance genes that can transpose onto resident/circulating plasmids; and (iii) is a site for the capture of further IS26-associated resistance gene cargo.

© 2016 Wiley Periodicals, Inc. Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat “hit and miss” with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217–228, 2018.

© 2018 Rychener, In-Albon, Djordjevic, Chowdhury, Nicholson, Ziech, de Vargas, Frey and Falquet. A corrigendum on Clostridium chauvoei, an Evolutionary Dead-End Pathogen by Rychener, L., InAlbon, S., Djordjevic, S. P., Chowdhury, P. R., Ziech, R. E., de Vargas, A. C., et al. (2017). Front. Microbiol. 8:1054. doi: 10.3389/fmicb.2017.01054. In the published article, Pamela Nicholson was not included as an author. In addition, author names were incorrectly spelled as Saria InAlbon and Piklu R. Chowdhury. The correct spellings are Saria In-Albon and Piklu Roy Chowdhury. The authors apologize for these errors and state that they do not affect the scientific conclusions of the article in any way. The original article has been updated. Author Contributions: JF, LF, and SD conceived the study and performed the genomic analyses. PN performed strain isolations and DNA preparations. Bioinformatic analyses was made by SI-A and LR. PC established the phylogenic relationship using by PhyloSift. RZ and AdV provided strains and metadata from strains of Brazil. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

© 2017 American Chemical Society. The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.

© 2018, American Society for Mass Spectrometry. Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time. [Figure not available: see fulltext.].

Copyright © 2015 John Wiley & Sons, Ltd. Rationale This paper, in conjunction with a work published earlier this year by O'Rourke et al., aims to provide a comprehensive set of protocols for the analysis of formalin-fixed paraffin-embedded (FFPE) tissue via matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) in a low-cost and highly repeatable and robust way, thereby allowing other research teams to begin their own IMS-centered avenues of research. Methods Samples of FFPE tissue were sectioned at 5 μm, water float mounted to specially prepared ITO glass slides and then dilipidated in a graded alcohol series. Tissue sections were then antigen retrieved under pressure in 20 mmol Tris-HCl (pH 8.8), coated with trypsin and digested O/N at 37°C. Samples were then sublimated with matrix to a final coverage of 0.2 mg/cm2, recrystallised at 37 C with 50:50 acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) for 1 h and analysed with a MALDI TOF/TOF mass spectrometer. Results Serial sections were imaged, revealing little to no variation with regards to image quality and corresponding spectra. We have also attempted to describe the processes that govern the various aspects of this protocol with respect to each step necessary to ensure reproducibility. Conclusions We are confident that this protocol in conjunction with the work published earlier by O'Rourke et al. provides the basis for a repeatable and robust protocol for the analysis of tissues from various sources via MALDI-IMS. The descriptions of key steps within allows for easy adoption of the protocol while allowing for desired modifications to be performed with minimal yet intuitive adjustment.

© 2015. Transposons flanked by direct copies of IS. 26 are important contributors to the evolution of multiple antibiotic resistance. Tn. 6029 and Tn. 6026 are examples of composite transposons that have become widely disseminated on small and large plasmids with different incompatibility markers in pathogenic and commensal Escherichia coli and various serovars of Salmonella enterica. Some of the plasmids that harbour these transposons also carry combinations of virulence genes. Recently, Tn. 6029 and Tn. 6026 and derivatives thereof have been found on chromosomal islands in both established and recently emerged pathogens. While Tn. 6029 and Tn. 6026 carry genes encoding resistance to older generation antibiotics, they also provide a scaffold for the introduction of genes encoding resistance to a wide variety of clinically relevant antibiotics that are mobilised by IS. 26. As a consequence, Tn. 6029 and Tn. 6026 or variants are likely to increasingly feature in complex resistance regions in multiple antibiotic resistant Enterobacteriaceae that threaten the health of humans and food production animals.

© 2015 Elsevier B.V. In Australia, outbreaks of clinical theileriosis caused by Theileria orientalis have been largely associated with the Ikeda genotype which can occur as a sole infection, or more commonly, as a mixture of genotypes. The most prevalent genotype, Chitose, frequently co-occurs with type Ikeda, however the role of this genotype in clinical disease has not been clearly established. Furthermore, the dynamics of individual genotypes in field infection of cattle have not been examined. In this study we developed quantitative PCR (qPCR) and genotyping methods to examine the role of the Chitose genotype in clinical disease and to investigate the temporal dynamics of T. orientalis Ikeda, Chitose and Buffeli genotypes in naïve animals introduced to a T. orientalis-endemic area. Analysis of the major piroplasm surface protein (MPSP) genes of Chitose isolates revealed the presence of two distinct phylogenetic clusters, Chitose A and Chitose B. A genotyping assay aimed at determining Chitose A/B allele frequency revealed that the Chitose A phylogenetic cluster is strongly associated with clinical disease but nearly always co-occurs with the Ikeda genotype. qPCR revealed that the Chitose genotype (particularly Chitose A), undergoes temporal switching in conjunction with the Ikeda genotype and contributes substantially to the overall parasite burden. The benign Buffeli genotype can also undergo temporal switching but levels of this genotype appear to remain low relative to the Ikeda and Chitose types. Interplay between vector and host immunological factors is presumed to be critical to the population dynamics observed in this study. Genotypic switching likely contributes to the persistence of T. orientalis in the host.

© 2015 John Wiley & Sons, Ltd. Rationale There are currently multiple methods available for the preparation of fresh frozen tissue samples for analysis via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS). Although these methods report excellent results, many are expensive automated approaches. With no published attempt to standardise less expensive manual processes, our work aims to provide a robust and repeatable method of sample preparation for MALDI-TOF-IMS that is applicable to a variety of tissue types, well explained, simple and cost effective. Methods Fresh frozen tissue was sectioned at 12 μm and mounted onto liquid nitrocellulose coated slides, washed in a graded alcohol series and then mounted into a modified sublimation apparatus. Matrix is deposited onto the slide to achieve a desired coating of 0.2 mg/cm2. Once coated, the slide is mounted into a custom-built vapor chamber and recrystallised with 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) for 1 h at 37C. The slide is then analysed using MALDI-IMS. Results We have successfully implemented this method for a host of tissue samples, including brain, liver, kidney and heart, with no variation in relative spectra or processing method required. When the protocol is followed correctly, sublimations and recrystallisations are highly predictable with limited variation between samples and a very low failure rate. Additional apparatuses can be easily constructed by following the included instructions, that perform as per specifications with no variation. Conclusions We believe that we have described a complete protocol for MALDI-IMS that is easy to use and highly reproducible. The lack of expensive commercially available equipment makes this process very cheap with a relatively low initial outlay and our hope is that more laboratories will begin IMS-based avenues of research based on the work we have performed.

© 2014 John Wiley & Sons Ltd. Summary: Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M.hyopneumoniae and fibronectin and show that M.hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.

We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen. © 2013 Biochemical Society.

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60 384 and P50 384. The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115 385) and 88 kDa (P88 385) and 27 kDa (P27 385) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide 752IQFELEPISLNV 763 denotes the C-terminus of P88 385 and defines the novel cleavage site 761L-N-V↓A-V- S 766 in Mhp385. P115 385, P88 385, P27 385, P60 384, and P50 384 were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60 384, P50 384, and P88 385. No primary function was attributed to P27 385; however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae. © 2012 American Chemical Society.

Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50P146, P40P146, and P85P146 that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence 672ATEF↓QQ677, consistent with a cleavage motif resembling S/T-X-F↓XD/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1P146-F3P146 that mimic P50P146, P40P146, and P85P146 were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146 generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. © 2012 Bogema et al.

Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60kDa (P60) and C-terminal 42kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K D~76nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K Ds of 26 and 33nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence. © 2011 Blackwell Publishing Ltd.

Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa preprotein (P135) that is cleaved into three fragments identified here as P45 683, P48 683, and P50 683. A peptide sequence (TTKF ↓ QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48 683 and P50 683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45 683, P48 683, and P50 683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1 683-F5 683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1 683-F5 683 also bound porcine epithelial cilia, and antisera to F2 683 and F5 683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45 683, P48 683, and P50 683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Salmonella enterica serovar Typhimurium phage type 9 isolates resistant to streptomycin and sulfonamide have been recovered from both bovine and human sources in Australia. This study aimed to identify the resistance genes and their location. Polymerase chain reaction was used to screen for resistance genes and sul2 (sulphonamide resistance) and strA and strB (streptomycin resistance) were detected. A small streptomycin and sulfonamide resistance plasmid carrying the three resistance genes was recovered from these isolates by transformation and was shown to be essentially identical to the small IncQ plasmid RSF1010. The sequences of one plasmid, pSRC15, and RSF1010 differed at only a few positions that may be errors in the older sequence. RSF1010 has been recovered from many species and in many countries since its first isolation in the early 1970s. We conclude that this plasmid has persisted unchanged in the environment for over 30 years. The antibiotic resistance gene cluster containing strA, strB, and sul2 genes has clearly arisen from other known entities by a combination of transposition and homologous recombination using a short segment of homology. This resistance gene cluster is now widely distributed in plasmids and genomic islands in a number of contexts. © Copyright 2010, Mary Ann Liebert, Inc. 2010.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, adheres to ciliated respiratory epithelia resulting in ciliostasis and epithelial cell death. The cilium adhesin P97 (Mhp183) contains two repeat regions, designated R1 and R2, that play key roles in adherence. Eight pentapeptide repeats in R1 are sufficient to bind porcine cilia; however, both R1 and R2 are needed to bind heparin. Mhp271, a paralogue of P97, is the only other M. hyopneumoniae protein to contain both R1 and R2 repeats. These repeats are arranged as a set of three pentapeptide repeats (designated R1A271), two decapeptide repeats (designated R2271), and a second set of six pentapeptide repeats (designated R1B271). To determine their function, recombinant proteins containing R1A271 (F1271) and R2271-R1B271 (F2271) were constructed and used in in vitro binding assays. F2271, but not F1271, bound heparin (KD = 8.1 ± 0.4 nM), fibronectin (KD = 174 ± 13 nM) and porcine cilia. Pre-incubation of F2271 with 100 μM heparin blocked cilium binding by ~69%. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry analysis identified Mhp271 as surface-exposed. Our data suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules. © 2010 Blackwell Publishing Ltd.

Conjugative IncHI2 plasmids carrying tetracycline, trimethoprim, and sulphonamide resistance genes were recovered from two multiply antibiotic resistant Salmonella enterica serovar Typhimurium isolates from Australian food-producing animals. Transposons related to the mercury resistance transposon Tn1696 were identified in both IncHI2 plasmids. These transposons contained an In4-type class 1 integron that carried a dfrA5 trimethoprim resistance gene cassette and the sul1 sulfonamide resistance gene. These integrons were located in the same position as In4 in Tn1696. The integron from one isolate includes a large transposon-like structure containing four IS26 and the strAB, sul2, blaTEM, and aphA1 genes conferring resistance to streptomycin, sulphonamides, ampicillin, kanamycin, and neomycin, respectively. This structure is flanked by an 8-bp duplication, but it includes both the aphA1-containing transposon Tn4352 and a transposon, Tn6029, carrying genes derived from RSF1010 and from Tn2. However, Tn4352 and Tn6029 overlap, sharing one IS26 copy. This suggests that they do not move by a standard transpositional mechanism. A circular intermediate, carrying only the region containing the resistance gene(s) and one of the IS26 bounding it, is proposed as an intermediate. © 2010 Mary Ann Liebert, Inc.

Enterohemorrhagic Escherichia coli (EHEC) strains are zoonotic pathogens responsible for a range of severe human disease. The repertoire of virulence determinants promoting EHEC disease is encoded on both the main chromosome and virulence plasmid. We examined a multiply antibiotic-resistant O26 EHEC strain for carriage of resistance genes on the virulence plasmid. The EHEC virulence plasmid containing a complex antibiotic-resistance gene locus, designated as pO26-CRL, was purified from EHEC O26:H- (patient with hemorrhagic colitis) and subjected to shotgun-sequencing and bioinformatic analysis. Determination of the 111,481-bp sequence of pO26-CRL revealed genes encoding a functional enterohemolysin operon (ehxCABD), STEC-specific extracellular serine protease (espP), putative EHEC adhesin (toxB), catalase/peroxidase (katP), and myristoyl transferase (msbB) involved in lipid A synthesis. A 22,609-bp Tn21 derivative is inserted within the conjugal transfer gene traC and encodes resistance to trimethoprim, streptomycin, sulfathiozole, kanamycin, neomycin, β-lactams, and mercuric chloride. Plasmid pO26-CRL is nonconjugative but is mobilizable. This is the first report of an EHEC virulence plasmid containing a complex antibiotic resistance locus, and raises the concern that antibiotic use will coselect for virulence determinants, leading to increased disease potential in both commensal and pathogenic E. coli populations. © FASEB.

The first Australian isolate of Salmonella enterica serovar Paratyphi B D-tartrate-utilizing (dT+) that is resistant to ampicillin, chloramphenicol, florfenicol, streptomycin, spectinomycin, sulfonamides, and tetracycline (ApCmFlSmSpSuTc) and contains SGI1 was isolated from a patient with gastroenteritis in early 1995. This is the earliest reported isolation globally. The incidence of infections caused by these SGI1-containing multiply antibiotic-resistant S. enterica serovar Paratyphi B dT+ strains increased during the next few years and occurred sporadically in all states of Australia. Several molecular criteria were used to show that the early isolates are very closely related to one another and to strains isolated during the following few years and in 2000 and 2003 from home aquariums and their owners. Early isolates from travelers returning from Indonesia shared the same features. Thus, they appear to represent a true clone arising from a single cell that acquired SGI1. Some minor differences in the resistance profiles and molecular profiles also were observed, indicating the ongoing evolution of the clone, and phage type differences were common, indicating that this is not a useful epidemiological marker over time. Three isolates from 1995, 1998, and 1999 contained a complete sul1 gene but were susceptible to sulfamethoxazole due to a point mutation that creates a premature termination codon. This SGI1 type was designated SGI1-R. The loss of resistance genes also was examined. When strains were grown for many generations in the absence of antibiotic selection, the loss of SGI1 was not detected. However, variants SGI1-C (resistance profile SmSpSu) and SGI1-B (resistant to ApSu), which had lost part of the integron, arose spontaneously, presumably via homologous recombination between duplications in the In104 complex integron. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2P97 (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M(IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO2 chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide 90VSELpSFR96 (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1P216, F2 P216, F3P216) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1P216, F2P216 and F3P216 adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain. © 2008 Blackwell Publishing Ltd.

Multiply antibiotic-resistant Salmonella enterica serovar Emek strains isolated in Australia and the United Kingdom had similar features, suggesting that they all belong to a single clone. These strains all contain SGI2 (formerly SGI1-J), an independently formed relative of Salmonella genomic island SGI1. In SGI2, the complex class 1 integron which includes all of the resistance genes is not located between tnpR (S027) and S044 as in SGI1 and SGI1 variants. Instead, tnpR was found to be adjacent to S044, and the integron is located 6.9 kb away, within S023. In both SGI1 and SGI2, the 25-bp inverted repeats that mark the outer ends of class 1 integrons are flanked by a 5-bp duplication of the target, indicating that incorporation of the integron was by transposition. A small number of differences between the sequences of the backbones of SGI1 and SGI2 were also found. Hence, a class 1 integron has entered two different variants of the SGI backbone to generate two distinct lineages. Despite this, the integron in SGI2 has a complex structure that is very similar to that of In104 in SGI1. Differences are in the cassette arrays and in the gene which encodes the chloramphenicol and florfenicol efflux protein. The CmlA9 protein, encoded by InEmek, is only 92.8% identical to FloRc (also a CmlA family protein) from SGI1. A variant form of SGI2, SGI2-A, which has lost the tet(G) and cmlA9 resistance determinants, was found in one strain. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5ΔhtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5ΔhtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5ΔhtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

Mycoplasma gallisepticum is an avian pathogen that causes a chronic respiratory disease of chickens and results in significant economic losses to the poultry industry worldwide. Colonization of the host and the establishment of chronic disease are initiated by the cytadherence of M. gallisepticum to the host respiratory epithelium. While several proteins involved in cytadhesion have been characterized, molecules that interact with components of the host extracellular matrix, a process that is central to pathogenesis, are only now being identified. In this study, M. gallisepticum whole cells were shown to bind heparin in a specific and saturable manner. Heparin also significantly inhibited the binding of M. gallisepticum to the human lung fibroblast cell line MRC-5, suggesting a potential role for glycosaminoglycans (GAGs) in cytadherence. M. gallisepticum protein MG1142 (encoded by mga_1142), which displays homology to the osmotically induced (OsmC) family of proteins, binds strongly to heparin, is highly expressed during in vitro culture, and is surface accessible. Recombinant MG1142 bound heparin in a dose-dependent and saturable manner with a dissociation constant (Kd) of 10 ± 1.8 nM, which is within a physiologically significant range, compared to that of other heparin-binding proteins. Binding to heparin was inhibited by the heavily sulfated polysaccharide fucoidan, but not by mucin or chondroitin sulfate A or B, suggesting that electrostatic interactions between the sulfate groups of heparin and the positively charged basic residues of the MG1142 protein are important in binding. The ability of M. gallisepticum to bind GAGs may contribute to host adherence and colonization. © 2007 SGM.

Multilocus sequence typing (MLST) has provided important new insights into the population structure of Campylobacter jejuni and is rapidly becoming the gold standard for typing this species. However, the methodology is comparatively costly and slow to perform for the routine surveillance testing of large numbers of isolates required by public health laboratories. Restriction fragment length polymorphism analysis of the flaA gene (RELP-flaA) and sequencing of the variable region in the fla locus (SVR-fla) were compared to MLST to determine if a low cost alternative could be found that reliably predicts clonal lineage (as determined by MLST). An isolate of C. jejuni from each of 153 patients from New South Wales, Australia, collected sequentially over a period of 30 months from 1999 to 2001 and comprising 40 sequence types (ST) from 15 clonal complexes (CC) was examined. Of 15 CC, 12 were represented by more than one isolate and a predominant RFLP-flaA type was found for 10 (83%). Of these, seven (70%) correctly predicted the predominant MLST CC with a probability of >0.8. Of 40 STs detected, 19 were reported for the first time, 9 of which were represented by more than one isolate. Eight of these were represented by a single RFLP-flaA type. Only two of eight major SVR-fla types were able to predict CC with a probability of >0.8, indicating that flaA-RFLP is a more reliable predictor of CC than SVR-fla and thus offers an alternative to MLST for use in routine surveillance. Copyright © 2007, American Society for Microbiology. All Rights Reserved.