Comparison of rectal swabs and fecal samples for the detection of Clostridioides difficile infections with a new in-house PCR assay.

The detection of Clostridioides difficile infections (CDI) relies on testing the stool of patients by toxin antigen detection or PCR methods. Although PCR and antigenic methods have significantly reduced the time to results, delays in stool collection can significantly add to the turnaround time. The use of rectal swabs to detect C. difficile could considerably reduce the time to diagnosis of CDI. We developed a new rapid PCR assay for the detection of C. difficile and evaluated this PCR assay on both stool and rectal swab specimens. We recruited a total of 623 patients suspected of C. difficile infection. Stool samples and rectal swabs were collected from each patient and tested by our PCR assay. Stool samples were also tested by the cell cytotoxicity neutralization assay (CCNA) as a reference. The PCR assay detected C. difficile in 60 stool specimens and 61 rectal swabs for the 64 patients whose stool samples were positive for C. difficile by CCNA. The PCR assay detected an additional 35 and 36 stool and rectal swab specimens positive for C. difficile, respectively, for sensitivity with stools and rectal swabs of 93.8% and 95.3%, specificity of 93.7% and 93.6%, positive predictive values of 63.2% and 62.9%, and negative predictive values of 99.2% and 99.4%. Detection of C. difficile using PCR on stools or rectal swabs yielded reliable and similar results. The use of PCR tests on rectal swabs could reduce turnaround time for CDI detection, thus improving CDI management and control of C. difficile transmission. IMPORTANCE: Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated diarrhea, resulting in high morbidity, mortality, and economic burden. In clinical laboratories, CDI testing is currently performed on stool samples collected from patients with diarrhea. However, the diagnosis of CDI can be delayed by the time required to collect stool samples. Barriers to sample collection could be overcome by using a rectal swab instead of a stool sample. Our study showed that CDI can be identified rapidly and reliably by a new PCR assay developed in our laboratory on both stool and rectal swab specimens. The use of PCR tests on rectal swabs could reduce the time for the detection of CDI and improve the management of this infection. It should also provide a useful alternative for infection-control practitioners to better control the spread of C. difficile.

Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention.

Infectious diseases (IDs) are a leading cause of death. The diversity and adaptability of microbes represent a continuing risk to health. Combining vision with passion, our transdisciplinary medical research team has been focussing its work on the better management of infectious diseases for saving human lives over the past five decades through medical discoveries and innovations that helped change the practice of medicine. The team used a multiple-faceted and integrated approach to control infectious diseases through fundamental discoveries and by developing innovative prevention tools and rapid molecular diagnostic tests to fulfill the various unmet needs of patients and health professionals in the field of ID. In this article, as objectives, we put in context two main research areas of ID management: innovative infection prevention that is woman-controlled, and the rapid molecular diagnosis of infection and resistance. We also explain how our transdisciplinary approach encompassing specialists from diverse fields ranging from biology to engineering was instrumental in achieving success. Furthermore, we discuss our vision of the future for translational research to better tackle IDs.

Characterization of vancomycin-resistance vanD gene clusters in the human intestinal microbiota by metagenomics and culture-enriched metagenomics.

Objectives: To characterize vancomycin-resistance vanD gene clusters and potential vanD-carrying bacteria in the intestinal microbiota of healthy volunteers exposed or not to ß-lactam antibiotics. Methods: Stool samples were collected before and after 7 days of cefprozil ß-lactam antibiotic exposure of 18 participants and six control participants who were not exposed to the antibiotic at the same time points. Metagenomic sequencing and culture-enriched metagenomic sequencing (with and without ß-lactam selection) were used to characterize vanD gene clusters and determine potential vanD-carrying bacteria. Alteration by antimicrobials was also examined. Results: Culture enrichment allowed detection of vanD genes in a large number of participants (11/24; 46%) compared to direct metagenomics (2/24; 8%). vanD genes were detected in stool cultures only following ß-lactam exposure, either after ß-lactam treatment of participants or after culture of stools with ß-lactam selection. Six types of vanD gene clusters were identified. Two types of vanD cluster highly similar to those of enterococci were found in two participants. Other vanD genes or vanD clusters were nearly identical to those identified in commensal anaerobic bacteria of the families Lachnospiraceae and Oscillospiraceae and/or bordered by genomic sequences similar or related to these anaerobes, suggesting that they are the origin or carriers of vanD. Conclusions: This study showed that culture-enriched metagenomics allowed detection of vanD genes not detected by direct metagenomics and revealed collateral enrichment of bacteria containing vancomycin-resistance vanD genes following exposure to ß-lactams, with a higher prevalence of the most likely gut commensal anaerobes carrying vanD. These commensal anaerobes could be the reservoir of vanD genes carried by enterococci.

Culture-enriched human gut microbiomes reveal core and accessory resistance genes.

BACKGROUND: Low-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes. Unfortunately, these less-abundant bacteria can be overlooked by deep shotgun sequencing. In addition, it is a challenge to associate the presence of resistance genes with their risk of acquisition by pathogens. In this study, we used liquid culture enrichment of stools to assemble the genome of lower-abundance bacteria from fecal samples. We then investigated the gene content recovered from these culture-enriched and culture-independent metagenomes in relation with their taxonomic origin, specifically antibiotic resistance genes. We finally used a pangenome approach to associate resistance genes with the core or accessory genome of Enterobacteriaceae and inferred their propensity to horizontal gene transfer. RESULTS: Using culture-enrichment approaches with stools allowed assembly of 187 bacterial species with an assembly size greater than 1 million nucleotides. Of these, 67 were found only in culture-enriched conditions, and 22 only in culture-independent microbiomes. These assembled metagenomes allowed the evaluation of the gene content of specific subcommunities of the gut microbiome. We observed that differentially distributed metabolic enzymes were associated with specific culture conditions and, for the most part, with specific taxa. Gene content differences between microbiomes, for example, antibiotic resistance, were for the most part not associated with metabolic enzymes, but with other functions. We used a pangenome approach to determine if the resistance genes found in Enterobacteriaceae, specifically E. cloacae or E. coli, were part of the core genome or of the accessory genome of this species. In our healthy volunteer cohort, we found that E. cloacae contigs harbored resistance genes that were part of the core genome of the species, while E. coli had a large accessory resistome proximal to mobile elements. CONCLUSION: Liquid culture of stools contributed to an improved functional and comparative genomics study of less-abundant gut bacteria, specifically those associated with antibiotic resistance. Defining whether a gene is part of the core genome of a species helped in interpreting the genomes recovered from culture-independent or culture-enriched microbiomes.

The initial state of the human gut microbiome determines its reshaping by antibiotics.

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase blaCfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.

Method for rapid and sensitive detection of Enterococcus sp. and Enterococcus faecalis/faecium cells in potable water samples.

We have developed a rapid and robust technological solution including a membrane filtration and dissolution method followed by a molecular enrichment and a real-time PCR assay, for detecting the presence of Enterococcus sp. or Enterococcus faecalis/faecium per 100 mL of water in less than 5 h and we compared it to Method 1600 on mEI agar in terms of specificity, sensitivity, and limit of detection. The mEI and the Enterococcus sp.-specific assay detected respectively 73 (64.0%) and 114 (100%) of the 114 enterococcal strains tested. None of the 150 non-enterococcal strains tested was detected by both methods with the exception of Tetragenococcus solitarius for the Enterococcus sp. assay. The multiplexed E. faecalis/faecium assay efficiently amplified DNA from 47 of 47 (100%) E. faecalis and 27 of 27 (100%) E. faecium strains tested respectively, whereas none of the 191 non-E. faecalis/faecium strains tested was detected. By simultaneously detecting the predominant fecal enterococcal species, the E. faecalis/faecium-specific assay allows a better distinction between enterococcal strains of fecal origin and those provided by the environment than Method 1600. Our procedure allows the detection of 4.5 enterococcal colony forming units (CFU) per 100 mL in less than 5 h, whereas the mEI method detected 2.3 CFU/100 mL in 24 h (95% confidence). Thus, our innovative and highly effective method provides a rapid and easy approach to concentrate very low numbers of enterococcal cells present in a 100 mL water sample and allows a better distinction between fecal and environmental enterococcal cells than Method 1600.

ampG gene of Pseudomonas aeruginosa and its role in ß-lactamase expression.

In enterobacteria, the ampG gene encodes a transmembrane protein (permease) that transports 1,6-GlcNAc-anhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampC ß-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosa carries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coli AmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosa strains PAO1 and PAK, inactivation of ampG drastically repressed the intrinsic ß-lactam resistance while ampGh1 deletion had little effect on the resistance. Further, deletion of ampG in an ampD-null mutant abolished the high-level ß-lactam resistance that is associated with the loss of AmpD activity. The cloned ampG gene is able to complement both the P. aeruginosa and the E. coli ampG mutants, while that of ampGh1 failed to do so, suggesting that PA4393 encodes the only functional AmpG protein in P. aeruginosa. We also demonstrate that the function of AmpG in laboratory strains of P. aeruginosa can effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to ß-lactams among laboratory as well as clinical isolates of P. aeruginosa. Our results suggest that inhibition of the AmpG activity is a potential strategy for enhancing the efficacy of ß-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpC-hyperproducing clinical isolates.

Analytical limits of three beta-glucosidase-based commercial culture methods used in environmental microbiology, to detect enterococci.

The enzyme-based test methods Enterolert, Chromocult Enterococci agar, and mEI agar, used to assess water quality through the detection Enterococcus spp., have been compared in terms of their analytical specificity and their ability to detect various enterococcal strains. To achieve this goal, we have tested 110 different non-enterococcal bacterial strains and 101 strains of Enterococcus spp. isolated from diverse origins. The results obtained showed that 69 (68.3%), 84 (83.2%), and 89 (88.1%) of the 101 enterococcal strains tested respectively yielded a positive signal with Enterolert, mEI, and Chromocult Enterococci. Regarding the specificity, none of the non-Enterococcus spp. strains tested were detectable by any of the three culture methods, except for Granulicatella adiacens which turned out positive on Chromocult Enterococci. The results of this study showed that, based on our collection of strains, the Enterolert test method detected less enterococcal strains than the two other methods.

Plastic polymers for efficient DNA microarray hybridization: application to microbiological diagnostics.

Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations.

Vancomycin-modified nanoparticles for efficient targeting and preconcentration of Gram-positive and Gram-negative bacteria.

A series of vancomycin-modified nanoparticles were developed and employed in magnetic confinement assays to isolate a variety of Gram-positive and Gram-negative bacteria from aqueous solution. We determined that the orientation/architecture of vancomycin on the surface of the nanoparticles and the overall surface coverage is critical in mediating fast and effective interactions between the nanoparticle and the pathogen cell wall surface and only one orientation/architecture in a series of modified nanoparticles leads to the efficient and reproducible capture of several important pathogenic bacteria. Interestingly, as the nanoparticles increase in diameter (from approximately 50 to 2800 nm), it is necessary to incorporate a long linker between the nanoparticle surface and the vancomycin moiety in order for the surface bound probe to efficiently confine Gram-positive bacteria. Finally, we also determined that the time required for efficient labeling and subsequent magnetic confinement significantly decreases as the size of the nanoparticle and the vancomycin surface coverage on the nanoparticle increases. As disease detection technologies transition to "lab-on-a-chip" based platforms it is necessary to develop strategies to effectively and inexpensively preconcentrate cells from large volume to volumes more amenable to these types of microfluidic devices. These small molecule-modified superparamagnetic nanoparticles can provide a means by which this can be accomplished.

A low-cost, disposable card for rapid polymerase chain reaction.

A low-cost, disposable card for rapid polymerase chain reaction (PCR) was developed in this work. Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated with structured polycarbonate films to form microreactors in a card format. Ice valves [1] were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and investigate the thermal gradient in the reaction chamber in the direction of sample thickness. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 copy of E. coli tuf gene in 27 min.

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid.

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.

Identification of methicillin-resistant Staphylococcus aureus carriage in less than 1 hour during a hospital surveillance program.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide and is responsible for significant morbidity, mortality, and health care costs. Control strategies to limit the emergence and spread of this organism rely on rapid and sensitive tests for detection of MRSA carriage. However, the standard surveillance culture method for detecting MRSA is labor intensive and time-consuming (2-3 days per procedure). There is thus a need for a rapid and accurate method to screen for MRSA carriage. METHODS: We recently developed an easy-to-use real-time polymerase chain reaction (PCR) assay suitable for specific detection of MRSA in nasal specimens in <1 h. We studied the efficacy of our new PCR assay in routine screening for nasal MRSA carriage during a hospital surveillance program. A total of 331 nasal specimens obtained from 162 patients at risk for colonization were tested by both the standard mannitol agar culture method and our PCR assay. RESULTS: The PCR assay detected MRSA in all 81 samples that were culture positive for MRSA. The PCR assay detected 4 additional MRSA-positive specimens, for a specificity of 98.4%, a positive predictive value of 95.3%, and a sensitivity and negative predictive value of 100%. CONCLUSIONS: This novel PCR assay allows reliable identification of MRSA carriers in <1 h. This test should facilitate the efficacy of MRSA surveillance programs.

Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species.

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.

Real-time PCR assay for detection of fluoroquinolone resistance associated with grlA mutations in Staphylococcus aureus.

Resistance to fluoroquinolones among clinical isolates of Staphylococcus aureus has become a clinical problem. Therefore, a rapid method to identify S. aureus and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of S. aureus and mutations at codons Ser-80 and Glu-84 of the grlA gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-S. aureus bacterial species tested. A total of 85 S. aureus isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the S. aureus isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of S. aureus and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of S. aureus and its susceptibility to fluoroquinolones.