Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

ABSTRACTThe pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates ofClostridium perfringenstype A. To determine the location and mobility of thenetBstructural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in whichnetBwas insertionally inactivated by the chloramphenicol and thiamphenicol resistance genecatP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and thenetBgene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 fromC. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained thenetBgene and other potential virulence genes, and (iii) a 70-kb plasmid that carried thecpb2gene, which encodes a different pore-forming toxin, beta2 toxin.IMPORTANCEThe anaerobic bacteriumClostridium perfringenscan cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries thenetBgene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids fromC. perfringens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is the first report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.

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Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01814-17 ◽

2017 ◽

Vol 83 (24) ◽

Cited By ~ 11

Author(s):

Jake A. Lacey ◽

Anthony L. Keyburn ◽

Mark E. Ford ◽

Ricardo W. Portela ◽

Priscilla A. Johanesen ◽

...

Keyword(s):

Antibiotic Resistance ◽

Clostridium Perfringens ◽

Plasmid Transfer ◽

Conjugative Plasmid ◽

Conjugative Transfer ◽

Necrotic Enteritis ◽

Content Type ◽

Gastrointestinal Pathogen

ABSTRACT Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment. IMPORTANCE Clostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.

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Functional Identification of Conjugation and Replication Regions of the Tetracycline Resistance Plasmid pCW3 from Clostridium perfringens

Journal of Bacteriology ◽

10.1128/jb.00298-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4942-4951 ◽

Cited By ~ 74

Author(s):

Trudi L. Bannam ◽

Wee Lin Teng ◽

Dieter Bulach ◽

Dena Lyras ◽

Julian I. Rood

Keyword(s):

Antibiotic Resistance ◽

Clostridium Perfringens ◽

Tetracycline Resistance ◽

Gastrointestinal Diseases ◽

Functional Identification ◽

Plasmid Evolution ◽

Virulence Plasmids ◽

Resistance Plasmid ◽

Replication Region ◽

Human Infections

ABSTRACT Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes’ genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.

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Entry Exclusion of Conjugative Plasmids of the IncA, IncC, and Related Untyped Incompatibility Groups

Journal of Bacteriology ◽

10.1128/jb.00731-18 ◽

2019 ◽

Vol 201 (10) ◽

Cited By ~ 10

Author(s):

Malika Humbert ◽

Kévin T. Huguet ◽

Frédéric Coulombe ◽

Vincent Burrus

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Phylogenetic Analyses ◽

Antibiotic Resistance Genes ◽

Genomic Islands ◽

Pathogenic Species ◽

Incompatibility Group ◽

Content Type ◽

Conjugative Plasmids ◽

Type Iv

ABSTRACTConjugative plasmids of incompatibility group C (IncC), formerly known as A/C2, disseminate antibiotic resistance genes globally in diverse pathogenic species ofGammaproteobacteria. Salmonellagenomic island 1 (SGI1) can be mobilized by IncC plasmids and was recently shown to reshape the conjugative type IV secretion system (T4SS) encoded by these plasmids to evade entry exclusion. Entry exclusion blocks DNA translocation between cells containing identical or highly similar plasmids. Here, we report that the protein encoded by the entry exclusion gene of IncC plasmids (eexC) mediates entry exclusion in recipient cells through recognition of the IncC-encoded TraGCprotein in donor cells. Phylogenetic analyses based on EexC and TraGChomologs predicted the existence of at least three different exclusion groups among IncC-related conjugative plasmids. Mating assays using Eex proteins encoded by representative IncC and IncA (former A/C1) and related untyped plasmids confirmed these predictions and showed that the IncC and IncA plasmids belong to the C exclusion group, thereby explaining their apparent incompatibility despite their compatible replicons. Representatives of the two other exclusion groups (D and E) are untyped conjugative plasmids found inAeromonassp. Finally, we determined through domain swapping that the carboxyl terminus of the EexC and EexE proteins controls the specificity of these exclusion groups. Together, these results unravel the role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion.IMPORTANCEIncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species ofGammaproteobacteriadue to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 ofSalmonella enterica. While historically grouped as “IncA/C,” IncA and IncC replicons were recently confirmed to be compatible and to abolish each other’s entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

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Draft Genome Sequence of Clostridium perfringens Strain TAMU, Which Causes Necrotic Enteritis in Broiler Chickens

Microbiology Resource Announcements ◽

10.1128/mra.01357-19 ◽

2020 ◽

Vol 9 (4) ◽

Author(s):

Catherine Ausland ◽

Adil Sabr Al-Ogaili ◽

Juan D. Latorre ◽

Guillermo Tellez-Isaias ◽

Billy M. Hargis ◽

...

Keyword(s):

Clostridium Perfringens ◽

Genome Sequence ◽

Broiler Chickens ◽

Draft Genome ◽

Gastrointestinal Diseases ◽

Intervention Strategies ◽

Draft Genome Sequence ◽

Necrotic Enteritis ◽

Content Type ◽

Deadly Disease

Clostridium perfringens causes severe gastrointestinal diseases, which include necrotic enteritis (NE) in chickens, a deadly disease worldwide. We report here the draft genome sequence of Clostridium perfringens strain TAMU, which was used in developing an NE chicken challenge model. This C. perfringens TAMU genome sequence will aid in advancing potential intervention strategies to reduce NE pathogenesis.

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Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

mBio ◽

10.1128/mbio.01744-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 39

Author(s):

Roy Ummels ◽

Abdallah M. Abdallah ◽

Vincent Kuiper ◽

Anouar Aâjoud ◽

Marion Sparrius ◽

...

Keyword(s):

Antibiotic Resistance ◽

Gene Transfer ◽

Horizontal Gene Transfer ◽

Antibiotic Resistance Genes ◽

Conjugative Plasmid ◽

Content Type ◽

Conjugative Plasmids ◽

Type Vii Secretion ◽

Type Iv ◽

Slow Growing

ABSTRACTConjugative plasmids have been identified in a wide variety of different bacteria, ranging from proteobacteria to firmicutes, and conjugation is one of the most efficient routes for horizontal gene transfer. The most widespread mechanism of plasmid conjugation relies on different variants of the type IV secretion pathway. Here, we describe the identification of a novel type of conjugative plasmid that seems to be unique for mycobacteria. Interestingly, while this plasmid is efficiently exchanged between different species of slow-growing mycobacteria, includingMycobacterium tuberculosis, it could not be transferred to any of the fast-growing mycobacteria tested. Genetic analysis of the conjugative plasmid showed the presence of a locus containing homologues of three type IV secretion system components and a relaxase. In addition, a new type VII secretion locus was present. Using transposon insertion mutagenesis, we show that in fact both these secretion systems are essential for conjugation, indicating that this plasmid represents a new class of conjugative plasmids requiring two secretion machineries. This plasmid could form a useful new tool to exchange or introduce DNA in slow-growing mycobacteria.IMPORTANCEConjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted thatM. tuberculosisdoes not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred toM. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of antibiotic resistance genes between pathogenic mycobacteria. The opportunity is that we could use this plasmid to generate new tools for the efficient introduction of foreign DNA in slow-growing mycobacteria.

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Alterations of Salmonella enterica Serovar Typhimurium Antibiotic Resistance under Environmental Pressure

Applied and Environmental Microbiology ◽

10.1128/aem.01173-18 ◽

2018 ◽

Vol 84 (19) ◽

Cited By ~ 14

Author(s):

Mengfei Peng ◽

Serajus Salaheen ◽

Robert L. Buchanan ◽

Debabrata Biswas

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Salmonella Enterica ◽

Tetracycline Resistance ◽

Multidrug Resistant ◽

Genetic Alterations ◽

Antibiotic Resistant ◽

Content Type ◽

Farm Systems ◽

Serovar Typhimurium

ABSTRACT Microbial horizontal gene transfer is a continuous process that shapes bacterial genomic adaptation to the environment and the composition of concurrent microbial ecology. This includes the potential impact of synthetic antibiotic utilization in farm animal production on overall antibiotic resistance issues; however, the mechanisms behind the evolution of microbial communities are not fully understood. We explored potential mechanisms by experimentally examining the relatedness of phylogenetic inference between multidrug-resistant Salmonella enterica serovar Typhimurium isolates and pathogenic Salmonella Typhimurium strains based on genome-wide single-nucleotide polymorphism (SNP) comparisons. Antibiotic-resistant S. Typhimurium isolates in a simulated farm environment barely lost their resistance, whereas sensitive S. Typhimurium isolates in soils gradually acquired higher tetracycline resistance under antibiotic pressure and manipulated differential expression of antibiotic-resistant genes. The expeditious development of antibiotic resistance and the ensuing genetic alterations in antimicrobial resistance genes in S. Typhimurium warrant effective actions to control the dissemination of Salmonella antibiotic resistance. IMPORTANCE Antibiotic resistance is attributed to the misuse or overuse of antibiotics in agriculture, and antibiotic resistance genes can also be transferred to bacteria under environmental stress. In this study, we report a unidirectional alteration in antibiotic resistance from susceptibility to increased resistance. Highly sensitive Salmonella enterica serovar Typhimurium isolates from organic farm systems quickly acquired tetracycline resistance under antibiotic pressure in simulated farm soil environments within 2 weeks, with expression of antibiotic resistance-related genes that was significantly upregulated. Conversely, originally resistant S. Typhimurium isolates from conventional farm systems lost little of their resistance when transferred to environments without antibiotic pressure. Additionally, multidrug-resistant S. Typhimurium isolates genetically shared relevancy with pathogenic S. Typhimurium isolates, whereas susceptible isolates clustered with nonpathogenic strains. These results provide detailed discussion and explanation about the genetic alterations and simultaneous acquisition of antibiotic resistance in S. Typhimurium in agricultural environments.

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The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽

10.1128/mbio.00338-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 24

Author(s):

Menglin Ma ◽

Jorge Vidal ◽

Juliann Saputo ◽

Bruce A. McClane ◽

Francisco Uzal

Keyword(s):

Clostridium Perfringens ◽

Regulatory System ◽

Gas Gangrene ◽

Necrotic Enteritis ◽

Vegetative Cells ◽

Content Type ◽

Two Component ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

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The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry

Infection and Immunity ◽

10.1128/iai.00975-16 ◽

2017 ◽

Vol 85 (6) ◽

Cited By ~ 15

Author(s):

Qiang Yu ◽

Dion Lepp ◽

Iman Mehdizadeh Gohari ◽

Tao Wu ◽

Hongzhuan Zhou ◽

...

Keyword(s):

Quorum Sensing ◽

Clostridium Perfringens ◽

Global Gene Expression ◽

Toxin Production ◽

Phospholipid Metabolism ◽

Necrotic Enteritis ◽

Content Type ◽

Protein Levels ◽

Global Gene Expression Analysis

ABSTRACT Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

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Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

mBio ◽

10.1128/mbio.00019-13 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 36

Author(s):

Xu-Xia Yan ◽

Corrine J. Porter ◽

Simon P. Hardy ◽

David Steer ◽

A. Ian Smith ◽

...

Keyword(s):

Lipid Bilayers ◽

Clostridium Perfringens ◽

Pore Formation ◽

Random Mutagenesis ◽

Planar Lipid Bilayers ◽

Necrotic Enteritis ◽

Channel Conductance ◽

Content Type ◽

Alpha Hemolysin ◽

Insight Into

ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

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Antibiotic Resistance among Clinical Ureaplasma Isolates Recovered from Neonates in England and Wales between 2007 and 2013

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00889-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 52-56 ◽

Cited By ~ 29

Author(s):

Michael L. Beeton ◽

Victoria J. Chalker ◽

Lucy C. Jones ◽

Nicola C. Maxwell ◽

O. Brad Spiller

Keyword(s):

Antibiotic Resistance ◽

Molecular Mechanisms ◽

Treatment Options ◽

Tetracycline Resistance ◽

Health Clinic ◽

Broth Microdilution ◽

England And Wales ◽

Content Type ◽

Microdilution Method ◽

Broth Microdilution Method

ABSTRACTUreaplasmaspp. are associated with numerous clinical sequelae with treatment options being limited due to patient and pathogen factors. This report examines the prevalence and mechanisms of antibiotic resistance among clinical strains isolated from 95 neonates, 32 women attending a sexual health clinic, and 3 patients under investigation for immunological disorders, between 2007 and 2013 in England and Wales. MICs were determined by using broth microdilution assays, and a subset of isolates were compared using the broth microdilution method and the Mycoplasma IST2 assay. The underlying molecular mechanisms for resistance were determined for all resistant isolates. Three isolates carried thetet(M) tetracycline resistance gene (2.3%; confidence interval [CI], 0.49 to 6.86%); two isolates were ciprofloxacin resistant (1.5%; CI, 0.07 to 5.79%) but sensitive to levofloxacin and moxifloxacin, while no resistance was seen to any macrolides tested. The MIC values for chloramphenicol were universally low (2 μg/ml), while inherently high-level MIC values for gentamicin were seen (44 to 66 μg/ml). The Mycoplasma IST2 assay identified a number of false positives for ciprofloxacin resistance, as the method does not conform to international testing guidelines. While antibiotic resistance amongUreaplasmaisolates remains low, continued surveillance is essential to monitor trends and threats from importation of resistant clones.

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