Antibiotic Resistance Plasmids Cointegrated into a Megaplasmid Harboring the blaOXA-427 Carbapenemase Gene.

OXA-427 is a new class D carbapenemase encountered in different species of Enterobacteriaceae in a Belgian hospital. To study the dispersal of this gene, we performed a comparative analysis of two plasmids containing the blaOXA-427 gene, isolated from a Klebsiella pneumoniae strain and an Enterobacter cloacae complex strain. The two IncA/C2 plasmids containing blaOXA-427 share the same backbone; in the K. pneumoniae strain, however, this plasmid is cointegrated into an IncFIb plasmid, forming a 321-kb megaplasmid with multiple multiresistance regions.

Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients.

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like α-hemolysin, and the α- and ß-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (SCC) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply that the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing.

Genetic features of livestock-associated Staphylococcus aureus ST9 isolates from Chinese pigs that carry the lsa(E) gene for quinupristin/dalfopristin resistance.

Whole-genome sequencing (WGS) was used to investigate the genetic features of the recently identified lsa(E) gene in porcine S. aureus ST9 isolates. Three quinupristin/dalfopristin-resistant isolates harboring the lsa(E) gene (two MRSA and one MSSA) were sequenced. Phylogenetic analysis of 184S. aureus genomes showed that ST9 porcine isolates belong to a distinct sequence cluster. Further analysis showed that all isolates were deficient in the recently described type IV restriction-modification system and SCCmec type XII was identified in the two MRSA isolates, which included a rare class C2 mec gene complex. A 24kb ΨSCC fragment was found in the MRSA and MSSA isolates sharing 99% nucleotide sequence homology with the ΨSCCJCSC6690 (O-2) element of a ST9 MRSA isolate from Thailand (accession number AB705453). Comparison of these ST9 isolates with 181 publically available S. aureus genomes identified 24 genes present in all (100%) ST9 isolates, that were absent from the most closely related human isolate. Our analysis suggests that the sequenced quinupristin/dalfopristin-resistant ST9 lineage represent a reservoir of mobile genetic elements associated with resistance and virulence features.

The Staphylococcal Cassette Chromosome mec type V from Staphylococcus aureus ST398 is packaged into bacteriophage capsids.

The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.

Distinction of Staphylococcal Cassette Chromosome mec type V elements from Staphylococcus aureus ST398.

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage.

High-resolution typing by MLVF unveils extensive heterogeneity of European livestock-associated methicillin-resistant Staphylococcus aureus isolates with the sequence type 398.

Methicillin-resistant Staphylococcus aureus sequence type 398 (MRSA ST398) has emerged in livestock worldwide. In particular, areas in Europe with high densities of livestock farming are affected. Consequently, the incidence of human colonization and infection with ST398 is rapidly increasing. Distinguishing different ST398 isolates with standard typing tools is problematic. The objective of this study was to examine the discriminatory power of Multiple-Locus Variable number tandem repeat Fingerprinting (MLVF) on a highly diverse ST398 collection. Our data show that MLVF combined with spa-typing is an attractive approach for high-resolution typing of ST398 isolates and unveiling their relatedness.

Microfluidic-chip-based multiple-locus variable-number tandem-repeat fingerprinting with new primer sets for methicillin-resistant Staphylococcus aureus.

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Requirement of the agr locus for colony spreading of Staphylococcus aureus.

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation.

Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2-DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post-transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.

Recombination between ccrC genes in a type V (5C2&5) staphylococcal cassette chromosome mec (SCCmec) of Staphylococcus aureus ST398 leads to conversion from methicillin resistance to methicillin susceptibility in vivo.

Various types of the staphylococcal cassette chromosome mec (SCCmec) are known to confer methicillin resistance on the human pathogen Staphylococcus aureus. Such cassettes are not always stably maintained. The present studies were aimed at identifying the mechanism underlying the in vivo conversion of methicillin-resistant S. aureus (MRSA) to methicillin-susceptible S. aureus (MSSA) derivatives as encountered in two patients suffering from pneumonia and an umbilicus infection, respectively. All MRSA and MSSA isolates identified belong to multilocus sequence type (MLST) 398, have spa type t034, and are Panton-Valentine leukocidin positive. Sequencing of 27,616 nucleotides from the chromosomal SCCmec insertion site in orfX to the hsdR gene for a restriction enzyme revealed a type V (5C2&5) SCCmec. Sequence comparisons show that parts of the cassette are highly similar to sequences within SCCmec elements from coagulase-negative staphylococci, indicating a possible common origin. The cassette investigated contains ccrC-carrying units on either side of its class C2b mec gene complex. In vivo loss of the mec gene complex was caused by recombination between the recombinase genes ccrC1 allele 8 and ccrC1 allele 10. In vitro, the SCCmec was very stable, and low-frequency MRSA-to-MSSA conversion was only observed when MRSA isolates were cultivated at 41 degrees C for prolonged periods of time. In this case also, loss of the mec complex was due to ccrC gene recombination. Interestingly, the MRSA and MSSA isolates studied displayed no detectable differences in competitive growth and virulence, suggesting that the presence of the intact type V (5C2&5) SCCmec has no negative bearing on staphylococcal fitness under the conditions used.

Corrigendum: Comprehensive Molecular Characterization of Escherichia coli Isolates from Urine Samples of Hospitalized Patients in Rio de Janeiro, Brazil.

[This corrects the article DOI: 10.3389/fmicb.2018.00243.].

Reduced Fitness Costs of mcr-1.2 Compared to Mutated pmrB in Isogenic Colistin-Resistant KPC-3-Producing Klebsiella pneumoniae.

In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-pmrBΔ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2-carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB, contributing to the success of this highly resistant hospital-adapted epidemiological lineage.IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2 An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.

Core/Whole Genome Multilocus Sequence Typing and Core Genome SNP-Based Typing of OXA-48-Producing Klebsiella pneumoniae Clinical Isolates From Spain.

Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48-producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur's BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0-10 and 0-14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches when using Institut Pasteur's BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, and 2-5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a previously established threshold for epidemiological link) was generally in concordance with a low number of allele differences (<8) when using the SeqSphere+ cgMLST scheme. The cgSNP analysis showed 6-29 SNPs in isolates with identical allelic SeqSphere+ cgMLST profiles and 16-61 cgSNPs among ST405 isolates. Furthermore, comparison of WGS-based typing results with previously obtained MLST and pulsed-field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics.

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics.

The emergence of nosocomial infections by multidrug-resistant Staphylococcus haemolyticus isolates has been reported in several European countries. Here, we report the first two complete genome sequences of S. haemolyticus sequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics.

From the wound to the bench: exoproteome interplay between wound-colonizing Staphylococcus aureus strains and co-existing bacteria.

Wound-colonizing microorganisms can form complex and dynamic polymicrobial communities where pathogens and commensals may co-exist, cooperate or compete with each other. The present study was aimed at identifying possible interactions between different bacteria isolated from the same chronic wound of a patient with the genetic blistering disease epidermolysis bullosa (EB). Specifically, this involved two different isolates of the human pathogen Staphylococcus aureus, and isolates of Bacillus thuringiensis and Klebsiella oxytoca. Particular focus was attributed to interactions of S. aureus with the two other species, because of the high staphylococcal prevalence among chronic wounds. Intriguingly, upon co-cultivation, none of the wound isolates inhibited each other's growth. Since the extracellular proteome of bacterial pathogens is a reservoir of virulence factors, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis and K. oxytoca were characterized by Mass Spectrometry to explore the inherent relationships between these co-exisiting bacteria. This revealed a massive reduction in the number of staphylococcal exoproteins upon co-culturing with K. oxytoca or B. thuringiensis. Interestingly, this decrease was particularly evident for extracellular proteins with a predicted cytoplasmic localization, which were recently implicated in staphylococcal virulence and epidemiology. Furthermore, our exoproteome analysis uncovered potential cooperativity between the two different S. aureus isolates. Altogether, the observed exoproteome variations upon co-culturing are indicative of unprecedented adaptive mechanisms that set limits to the production of secreted staphylococcal virulence factors.

Expansion of KPC-producing Klebsiella pneumoniae with various mgrB mutations giving rise to colistin resistance: the role of ISL3 on plasmids.

mcr-1 has been reported as the first plasmid-encoded gene conferring colistin resistance. In KPC-producing Klebsiella pneumoniae (KPC-KP), however, colistin resistance is rapidly emerging through other mechanisms. Resistance is frequently due to disruption of the mgrB gene by insertion sequences, e.g. ISL3. The aim of this study was to investigate the expansion of mgrB-mutated KPC-KP isolates. In addition, the localisation and targets of ISL3 sequences within the core and accessory genome of common KPC-KP lineages were identified. A total of 29 clinical K. pneumoniae isolates collected from Italian patients were randomly selected. Whole genome sequences were analysed for resistance genes, plasmids and insertion sequences. In addition, 27 colistin-resistant KPC-KP isolates from a previous study from Crete (Greece) were assessed. Clonal expansion of KPC-KP isolates with various mutations in mgrB among all lineages was observed. In two Italian MLST ST512 isolates and eight Greek ST258 isolates, an identical copy of ISL3 was inserted in mgrB nucleotide position 133. ISL3, a transposable restriction-modification system of 8154 nucleotides, was located on pKpQIL-like plasmids and may transpose into the chromosome. In four isolates, chromosomal integration of ISL3 in diverse inner membrane proteins other than mgrB was identified. Colistin resistance is most often explained by clonal expansion of isolates with mutated mgrB. pKpQIL-like plasmids, which are omnipresent in KPC-KP, carry insertion sequences such as ISL3 that have mgrB as a target hotspot for transposition. Transposition of insertion sequences from plasmids and subsequent clonal expansion may contribute to the emerging colistin resistance in KPC-KP.

There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis is one of the major periodontal agents and it has been recently hailed as a potential cause of the autoimmune disease rheumatoid arthritis. In particular, the peptidylarginine deiminase enzyme of P. gingivalis (PPAD) has been implicated in the citrullination of certain host proteins and the subsequent appearance of antibodies against citrullinated proteins, which might play a role in the etiology of rheumatoid arthritis. The aim of this study was to investigate the extracellular localization of PPAD in a large panel of clinical P. gingivalis isolates. Here we show that all isolates produced PPAD. In most cases PPAD was abundantly present in secreted outer membrane vesicles (OMVs) that are massively produced by P. gingivalis, and to minor extent in a soluble secreted state. Interestingly, a small subset of clinical isolates showed drastically reduced levels of the OMV-bound PPAD and secreted most of this enzyme in the soluble state. The latter phenotype is strictly associated with a lysine residue at position 373 in PPAD, implicating the more common glutamine residue at this position in PPAD association with OMVs. Further, one isolate displayed severely restricted vesiculation. Together, our findings show for the first time that neither the major association of PPAD with vesicles, nor P. gingivalis vesiculation per se, are needed for P. gingivalis interactions with the human host.

Comprehensive Molecular Characterization of Escherichia coli Isolates from Urine Samples of Hospitalized Patients in Rio de Janeiro, Brazil.

Urinary tract infections (UTIs) are often caused by Escherichia coli. Their increasing resistance to broad-spectrum antibiotics challenges the treatment of UTIs. Whereas, E. coli ST131 is often multidrug resistant (MDR), ST69 remains susceptible to antibiotics such as cephalosporins. Both STs are commonly linked to community and nosocomial infections. E. coli phylogenetic groups B2 and D are associated with virulence and resistance profiles making them more pathogenic. Little is known about the population structure of E. coli isolates obtained from urine samples of hospitalized patients in Brazil. Therefore, we characterized E. coli isolated from urine samples of patients hospitalized at the university and three private hospitals in Rio de Janeiro, using whole genome sequencing. A high prevalence of E. coli ST131 and ST69 was found, but other lineages, namely ST73, ST648, ST405, and ST10 were also detected. Interestingly, isolates could be divided into two groups based on their antibiotic susceptibility. Isolates belonging to ST131, ST648, and ST405 showed a high resistance rate to all antibiotic classes tested, whereas isolates belonging to ST10, ST73, ST69 were in general susceptible to the antibiotics tested. Additionally, most ST69 isolates, normally resistant to aminoglycosides, were susceptible to this antibiotic in our population. The majority of ST131 isolates were ESBL-producing and belonged to serotype O25:H4 and the H30-R subclone. Previous studies showed that this subclone is often associated with more complicated UTIs, most likely due to their high resistance rate to different antibiotic classes. Sequenced isolates could be classified into five phylogenetic groups of which B2, D, and F showed higher resistance rates than groups A and B1. No significant difference for the predicted virulence genes scores was found for isolates belonging to ST131, ST648, ST405, and ST69. In contrast, the phylogenetic groups B2, D and F showed a higher predictive virulence score compared to phylogenetic groups A and B1. In conclusion, despite the diversity of E. coli isolates causing UTIs, clonal groups O25:H4-B2-ST131 H30-R, O1:H6-B2-ST648, and O102:H6-D-ST405 were the most prevalent. The emergence of highly virulent and MDR E. coli in Brazil is of high concern and requires more attention from the health authorities.

Reprint of "Application of next generation sequencing in clinical microbiology and infection prevention".

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.