Closed genome sequence of Clostridium botulinum type B1 strain isolated from an infant botulism case in the United States.

We present the closed genome sequence of the Clostridium botulinum BT-22100019 strain isolated from the stool specimen of an infant diagnosed with botulism. With 4.33-Mb genome size and 28.0% G + C content, the bont/B1 gene encoded for botulinum neurotoxin serotype B was found on a 262 kb plasmid arranged in a ha+ orfx - cluster.

bettercallsal: better calling of Salmonella serotypes from enrichment cultures using shotgun metagenomic profiling and its application in an outbreak setting.

Most current Salmonella subtyping analyses rely on whole genome sequencing (WGS), which focuses on the high-resolution analysis of single genomes or multiple single genomes from the isolated colonies on microbiological agar plates. In this study, we introduce bioinformatics innovations for a metagenomic outbreak response workflow that accurately identifies multiple Salmonella serovars at the same time. bettercallsal is one of the first analysis tools to identify multiple Salmonella enterica serotypes from metagenomic or quasi-metagenomic datasets with high accuracy, allowing these isolate-independent methods to be incorporated into surveillance and root cause investigations. It was tested on an in silico benchmark dataset comprising 29 unique Salmonella serovars, 46 non-Salmonella bacterial genomes, and 10 viral genomes at varying read depths and on previously well-characterized and sequenced non-selective primary and selective enrichments of papaya and peach samples from separate outbreak investigations that resulted in the identification of multiple Salmonella serovars using traditional isolate culturing and WGS as well as nucleic acid assays. Analyses were also conducted on these datasets using a custom-built k-mer tool, SeqSero2, and Kallisto to compare serotype calling to bettercallsal. The in silico dataset analyzed with bettercallsal achieved the maximum precision, recall, and accuracy of 100, 83, and 94%, respectively. In the papaya outbreak samples, bettercallsal identified the presence of multiple serovars in agreement with the Luminex® xMAP assay results and also identified more serovars per sample, as evidenced by NCBI SNP clustering. In peach outbreak samples, bettercallsal identified two serovars in concordance with k-mer analysis and the Luminex xMAP assay. The genome hit reported by bettercallsal clustered with the chicken isolate genome, as reported by the FDA peach outbreak investigation from sequenced isolates (WGS). Overall, bettercallsal outperformed k-mer, Seqsero2, and Kallisto in identifying multiple serovars from enrichment cultures using shotgun metagenomic sequencing.

qPCR detection of viable Bacillus cereus group cells in cosmetic products.

Reference methods for microbiological safety assessments of cosmetics rely on culture methods that reveal colonies of live microorganisms on growth media. Rapid molecular technologies, such as qPCR, detects the presence of target DNA in samples from dead and viable cells. DNA intercalating dyes, such as propidium monoazide (PMAxx), are capable of restricting PCR amplification to viable microbial cells. Here we developed singleplex and multiplex real time (qPCR) assays for the detection of Bacillus cereus (B. cereus) using 16S rRNA and phosphatidylcholine-specific phospholipase C (PLC) gene specific sequences coupled with PMAxx. The limit of detection was determined to be ~ 1 log CFU/ml for 16S rRNA and 3 log CFU/ml for PLC detection in pure culture using an eye shadow isolate, B. cereus 3A. We assessed the inclusivity and exclusivity of our qPCR assays using 212 strains, including 143 members of B. cereus, 38 non- B. cereus. and 31 non-Bacillus species; inclusivity was 100% for the 16S rRNA and 97.9% for the PLC targets; the exclusivity was 100% for 16S rRNA and 98.6% for PLC targets. These qPCR assays were then used to assess samples of commercial cosmetics: one set of liquid face toners (N = 3), artificially contaminated with B. cereus 3A, and one set of powdered cosmetics (N = 8), previously determined to be contaminated with B. cereus. For some samples, test portions were analyzed by qPCR in parallel, with and without PMAxx treatment. All test portions were simultaneously streaked on BACARA plates to confirm viable cells of B. cereus, according to the culture method. We found no difference in sensitivity between the singleplex and the multiplex qPCR assays (P > 0.05). Inoculated samples that did not recover B. cereus on plates still showed amplification of the DNA targets. However, that amplification was significantly delayed in PMAxx -treated samples (P < 0.0001) with CT value differences of 7.82 for 16S rRNA and 7.22 for PLC. Likewise, amplification delay was significant (P < 0.0001) with inoculated samples that recovered B. cereus on plates with CT value differences of 2.96 and 2.36 for 16S rRNA and PLC, respectively, demonstrating the presence of dead cells in the samples. All our qPCR results correlated with detection on BACARA plates (kappa, k = 0.99), independently of the presence of PMAxx in the PCR assays. Nevertheless, the amplification threshold with PMAxx dyes was significantly higher than the non-PMAxx dyes. Our findings confirm qPCR can be used for more rapid detection of microorganisms in cosmetics, including B. cereus, and selective detection of viable cells can be improved using PMAxx dyes.

Genomic characterization of Bacillus cereus sensu stricto 3A ES isolated from eye shadow cosmetic products.

BACKGROUND: The Bacillus cereus group, also known as B. cereus sensu lato (s.l.) contains ubiquitous spore-forming bacteria found in the environment including strains from the B. cereus sensu stricto (s.s.) species. They occur naturally in a wide range of raw materials and in consumer products. Characterizing isolates that have survived in consumer products allows us to better understand the mechanisms that permit spores to persist and potentially cause illness. Here we characterize the draft genome sequence of B. cereus s. s. 3A-ES, originally isolated from eye shadow and since investigated in several cosmetic studies and compared it to other top ten published complete genome sequences of B. cereus s.l. members. RESULTS: The draft genome sequence of B. cereus s.s. 3A ES consisted of an average of 90 contigs comprising approximately 5,335,727 bp and a GC content of 34,988%, and with 5509 predicted coding sequences. Based on the annotation statistics and comparison to other genomes within the same species archived in the Pathosystems Resource Integration Center (PATRIC), this genome "was of good quality. Annotation of B. cereus s.s. 3A ES revealed a variety of subsystem features, virulence factors and antibiotic resistant genes. The phylogenetic analysis of ten B. cereus group members showed B. cereus s.s. 3A-ES to be a closely related homolog of B. cereus s.s. ATCC 14,579, an established reference strain that is not adapted for cosmetic microbiological studies. Survival of 3A-ES in eye shadow could be linked to predicted stress-response genes and strengthened by additional stress-response genes such as VanB-type, VanRB, CAT15/16, BcrA, BcrB, Lsa(B), and recA that are lacking in B. cereus s.s. ATCC 14,579. CONCLUSION: Our genomic analysis of B. cereus s.s. 3A-ES revealed genes, which may allow this bacterium to withstand the action of preservatives and inhibitors in cosmetics, as well as virulence factors that could contribute to its pathogenicity. Having a well-characterized strain obtained from eye-shadow may be useful for establishing a reference strain for cosmetics testing.

Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp. Enterica serovar Gaminera, S. Mbandaka, S. Tennesse, S. Minnesota, S. Senftenberg and S. Typhimurium. These new strains fluoresce when irradiated with UV light and maintain this phenotype in absence of antibiotic selection. Recombinants were phenotypically equivalent to the parent strain, except for S. Tennessee Sal66 that appeared Lac- on Xylose Lysine Deoxycholate (XLD) agar plates and Lac+ on Mac Conkey and Hektoen Enteric agar plates. Analysis of closed whole genome sequences revealed that Sal66 had lost one lactose operon; slower rates of lactose metabolism may affect lactose fermentation on XLD agar. These fluorescent enteric control strains were challenging to develop and should provide an easy and effective means of identifying cross-contamination.

Corrigendum: Phylogenetic Analyses of Shigella and Enteroinvasive Escherichia coli for the Identification of Molecular Epidemiological Markers: Whole-Genome Comparative Analysis Does Not Support Distinct Genera Designation.

[This corrects the article on p. 1573 in vol. 6, PMID: 26834722.].

The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood.

Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes(the group A Streptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the frulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-D-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment.

Complete Genome Sequence of Enteroinvasive Escherichia coli O96:H19 Associated with a Severe Foodborne Outbreak.

We present here the complete genome sequence of a strain of enteroinvasive Escherichia coli O96:H19 from a severe foodborne outbreak in a canteen in Italy in 2014. The complete genome may provide important information about the acquired pathogenicity of this strain and the transition between commensal and pathogenic E. coli.

Identification of five Shiga toxin-producing Escherichia coli genes by Luminex microbead-based suspension array.

To rapidly identify the presence of potentially virulent O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC), a PCR-based Luminex suspension assay was developed to detect the genes coding for four virulence factors (stx1, stx2, eae, and ehxA) plus the O157:H7-specific +93 uidA single nucleotide polymorphism.

Phylogenetic Analyses of Shigella and Enteroinvasive Escherichia coli for the Identification of Molecular Epidemiological Markers: Whole-Genome Comparative Analysis Does Not Support Distinct Genera Designation.

As a leading cause of bacterial dysentery, Shigella represents a significant threat to public health and food safety. Related, but often overlooked, enteroinvasive Escherichia coli (EIEC) can also cause dysentery. Current typing methods have limited ability to identify and differentiate between these pathogens despite the need for rapid and accurate identification of pathogens for clinical treatment and outbreak response. We present a comprehensive phylogeny of Shigella and EIEC using whole genome sequencing of 169 samples, constituting unparalleled strain diversity, and observe a lack of monophyly between Shigella and EIEC and among Shigella taxonomic groups. The evolutionary relationships in the phylogeny are supported by analyses of population structure and hierarchical clustering patterns of translated gene homolog abundance. Lastly, we identified a panel of 404 single nucleotide polymorphism (SNP) markers specific to each phylogenetic cluster for more accurate identification of Shigella and EIEC. Our findings show that Shigella and EIEC are not distinct evolutionary groups within the E. coli genus and, thus, EIEC as a group is not the ancestor to Shigella. The multiple analyses presented provide evidence for reconsidering the taxonomic placement of Shigella. The SNP markers offer more discriminatory power to molecular epidemiological typing methods involving these bacterial pathogens.

Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR.

Escherichia coli serogroup O157 is the pathogen most commonly associated with foodborne disease outbreaks, but epidemiological studies suggest that non-O157 Shiga toxin-producing E. coli (STEC) is a major player as well. The ten most clinically relevant STECs belong to serogroups O26, O103, O111, O145, O157, O91, O113, O128, O45, and O121; but emerging strains, such as O104:H4 that was identified with the 2011 German outbreak, could become more prevalent in the future. A 75-min conventional multiplex PCR assay, IS-5P, targeting the four virulence factors stx1, stx2, eae, and ehxA plus the O157:H7-specific +93 uidA single nucleotide polymorphism was developed to better assess the potential pathogenicity of STEC isolates. All 212 STEC DNAs showed one to five amplification products, while the non-E. coli DNA did not react to this multiplex PCR assay. Enrichment broths obtained from baby spinach, alfalfa sprouts, and cilantro artificially inoculated with O26, O103, and O121 STECs reacted positively to the multiplex assay. Unlike the current FDA BAM 5P PCR, designed for the specific detection of O157:H7, IS-5P will identify potentially harmful O157:H7 and non-O157 STECs so they can be removed from the nation's food supply.

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen.

Rapid identification and differentiation of non-O157 Shiga toxin-producing Escherichia coli using polymerase chain reaction coupled to electrospray ionization mass spectrometry.

A polymerase chain reaction (PCR)-mass spectroscopy assay was developed to identify non-O157 Shiga toxin-producing Escherichia coli (STEC) with Plex-ID biosensor system, a platform identifying short PCR amplicons by specific base compositions. This assay simultaneously amplifies five fragments of two housekeeping genes, two subunits of stx2 gene, and four other virulence genes of STEC. A total of 164 well-characterized STEC isolates were examined with the assay to build a DNA base composition database. Another panel of 108 diverse STEC isolates was tested with the established database to evaluate the assay's identification capability. Among the 108 isolates, the assay specificity was 100% for three (stx1, eae, and aggA) out of five tested virulence genes, but 99% for stx2 and 96% for hlyA, respectively. Main stx1/stx2 subtypes and multiple alleles of stx1/stx2 could be differentiated. The assay successfully identified several clinically significant serotypes, including O91:H14, O103:H25, O145:H28/NM, O113:H21, and O104:H4. Meanwhile, it was able to group isolates with different levels of pathogenic potential. The results suggest that this high-throughput method may be useful in clinical and regulatory laboratories for STEC identification, particularly strains with increased pathogenic potential.

Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.

UNLABELLED: Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-L-[methyl-(14)C]methionine with an apparent K(m) of 5.9 µM and a V(max) of 32 pmol min(-1) mg(-1). Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target. IMPORTANCE: Obligate intracellular parasites like Chlamydia have followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions in Chlamydia. The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation of Chlamydia to an obligate intracellular lifestyle, the specific and central roles of SAMHT in Chlamydia metabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.

Impact of azithromycin resistance mutations on the virulence and fitness of Chlamydia caviae in guinea pigs.

Azithromycin (AZM) is a major drug used in the treatment and prophylaxis of infections caused by Chlamydia, yet no significant clinical resistance has been reported for these obligate intracellular bacteria. Nevertheless, spontaneous AZM resistance (Azm(r)) arose in vitro at frequencies ranging from 3 x 10(-8) to 8 x 10(-10) for clonal isolates of Chlamydia caviae, which is a natural pathogen of guinea pigs. Sequencing of the unique 23S rRNA gene copy in 44 independent Azm(r) isolates identified single mutations at position A(2058) or A(2059) (Escherichia coli numbering system). While SP(6)AZ(1) (A(2058)C) and SP(6)AZ(2) (A(2059)C) Azm(r) mutants showed growth defects in cell culture and were less pathogenic in the guinea pig ocular infection model than in the parent SP(6), the three isogenic C. caviae isolates grew equally well in the animal. On the other hand, coinoculation of the C. caviae parent strain with one of the Azm(r) strains was detrimental for the mutant strain. This apparent lack of association between pathology and bacterial load in vivo showed that virulence of the two Azm(r) mutants of C. caviae was attenuated. While chlamydial growth in vitro reflects the ability of the bacteria to multiply in permissive cells, survival in the host is a balance between cellular multiplication and clearance by the host immune system. The obligate intracellular nature of Chlamydia may therefore limit emergence of resistance in vivo due to the strength of the immune response induced by the wild-type antibiotic-sensitive bacteria at the time of antibiotic treatment.

The chlamydial functional homolog of KsgA confers kasugamycin sensitivity to Chlamydia trachomatis and impacts bacterial fitness.

BACKGROUND: rRNA adenine dimethyltransferases, represented by the Escherichia coli KsgA protein, are highly conserved phylogenetically and are generally not essential for growth. They are responsible for the post-transcriptional transfer of two methyl groups to two universally conserved adenosines located near the 3'end of the small subunit rRNA and participate in ribosome maturation. All sequenced genomes of Chlamydia reveal a ksgA homolog in each species, including C. trachomatis. Yet absence of a S-adenosyl-methionine synthetase in Chlamydia, the conserved enzyme involved in the synthesis of the methyl donor S-adenosyl-L-methionine, raises a doubt concerning the activity of the KsgA homolog in these organisms. RESULTS: Lack of the dimethylated adenosines following ksgA inactivation confers resistance to kasugamycin (KSM) in E. coli. Expression of the C. trachomatis L2 KsgA ortholog restored KSM sensitivity to the E. coli ksgA mutant, suggesting that the chlamydial KsgA homolog has specific rRNA dimethylase activity. C. trachomatis growth was sensitive to KSM and we were able to isolate a KSM resistant mutant of C. trachomatis containing a frameshift mutation in ksgA, which led to the formation of a shorter protein with no activity. Growth of the C. trachomatis ksgA mutant was negatively affected in cell culture highlighting the importance of the methylase in the development of these obligate intracellular and as yet genetically intractable pathogens. CONCLUSION: The presence of a functional rRNA dimethylase enzyme belonging to the KsgA family in Chlamydia presents an excellent chemotherapeutic target with real potential. It also confirms the existence of S-adenosyl-methionine--dependent methylation reactions in Chlamydia raising the question of how these organisms acquire this cofactor.

Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation.

To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 x 10(-9)). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 microg of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 +/- 1.1 x 10(-6), n = 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia.

Frequency of development and associated physiological cost of azithromycin resistance in Chlamydia psittaci 6BC and C. trachomatis L2.

Azithromycin is a major drug used in the treatment and prophylaxis of chlamydial infections. Spontaneous azithromycin-resistant mutants of Chlamydia psittaci 6BC were isolated in vitro in the plaque assay at a frequency of about 10(-8). Isogenic clonal variants with A(2058)C, A(2059)G, or A(2059)C mutations in the unique 23S rRNA gene (Escherichia coli numbering system) displayed MICs for multiple macrolides (i.e., azithromycin, erythromycin, josamycin, and spiramycin) at least 100 times higher than those of the parent strain and were also more resistant to the lincosamide clindamycin. Chlamydia trachomatis L2 variants with a Gln-to-Lys substitution in ribosomal protein L4 at position 66 (E. coli numbering system), conferring an eightfold decrease in azithromycin and erythromycin sensitivities and a fourfold decrease in josamycin and spiramycin sensitivities, were isolated following serial passage in subinhibitory concentrations of azithromycin. Each mutation was stably maintained in the absence of selection but severely affected chlamydial infectivity, as determined by monitoring the development of each isolate over 46 h in the absence of selection, in pure culture or in 1:1 competition with the isogenic parent. Data in this study support the hypothesis that the mechanisms which confer high-level macrolide resistance in chlamydiae carry a prohibitive physiological cost and may thus limit the emergence of highly resistant clones of these important pathogens in vivo.

Fitness cost due to mutations in the 16S rRNA associated with spectinomycin resistance in Chlamydia psittaci 6BC.

The fitness cost of a resistance determinant is the primary parameter that determines its frequency in vivo. As a model for analysis of the impact of drug resistance mutations on the intracellular life cycle of Chlamydia spp., we studied the growth of four genetically defined spectinomycin-resistant (Spc(r)) clonal variants of Chlamydia psittaci 6BC isolated in the plaque assay. The development of each variant was monitored over 46 h postinfection in the absence of drug, either in pure culture or in 1:1 competition with the parent strain. Spc(r) mutations in the 16S rRNA gene at positions 1191 and 1193 were associated with a marked impairment of C.psittaci biological fitness, and the bacteria were severely out-competed by the wild-type parent. In contrast, mutations at position 1192 had minor effects on the bacterial life cycle, allowing the resistant isolates to compete more efficiently with the wild-type strain. Thus, mutations with a wide range of fitness costs can be selected in the plaque assay, providing a new strategy for prediction and monitoring of the emergence of antibiotic resistance in chlamydiae. So far, drug resistance has not been a serious threat for the treatment of chlamydial infections. Tetracycline is an effective antichlamydial drug that targets 16S rRNA. Attempts to isolate spontaneous tetracycline-resistant mutants of C. psittaci 6BC revealed a frequency <3 x 10(-9). We suggest that the rarity of genotypic antibiotic resistance among chlamydial clinical isolates reflects the deleterious effects of such mutations on the fitness of these obligate intracellular bacteria in the host.

Frequency of spontaneous mutations that confer antibiotic resistance in Chlamydia spp.

Mutations in rRNA genes (rrn) that confer resistance to ribosomal inhibitors are typically recessive or weakly codominant and have been mostly reported for clinical strains of pathogens possessing only one or two rrn operons, such as Helicobacter pylori and Mycobacterium spp. An analysis of the genome sequences of several members of the Chlamydiaceae revealed that these obligate intracellular bacteria harbor only one or two sets of rRNA genes. To study the contribution of rRNA mutations to the emergence of drug resistance in the Chlamydiaceae, we used the sensitivities of Chlamydia trachomatis L2 (two rrn operons) and Chlamydophila psittaci 6BC (one rrn operon) to the aminoglycoside spectinomycin as a model. Confluent cell monolayers were infected in a plaque assay with about 10(8) wild-type infectious particles and then treated with the antibiotic. After a 2-week incubation time, plaques formed by spontaneous spectinomycin-resistant (Spc(r)) mutants appeared with a frequency of 5 x 10(-5) for C. psittaci 6BC. No Spc(r) mutants were isolated for C. trachomatis L2, although the frequencies of rifampin resistance were in the same range for both strains (i.e., 10(-7)). The risk of emergence of Chlamydia strains resistant to tetracyclines and macrolides, the ribosomal drugs currently used to treat chlamydial infections, is discussed.