Development and standardization of a specific real-time PCR assay for the rapid detection of Candida auris.

Candida auris is an emerging multiresistant pathogen causing nosocomial fungal infection. Specific detection and identification are necessary. Our goal is to develop a new qPCR system that enables rapid detection of C. auris, based on a GPI (glycosyl-phosphatidylinositol) protein-encoding gene. This system is reproducible and sensitive with a limit of detection of 13 C. auris CFU/qPCR reaction. The 100% specificity of this system is confirmed on 2073 clinical and environmental samples, 50 different bacterial species, and 9 Candida spp. (70 strains). This system is suitable to correctly identify C. auris infections and to trace its source.

Detection of plasmid-mediated colistin resistance, mcr-1 gene, in Escherichia coli isolated from high-risk patients with acute leukemia in Spain.

BACKGROUND: Bacterial infections in immunocompromised patients are associated with a high mortality and morbidity rate. In this high-risk group, the presence of multidrug-resistant (MDR) bacteria, particularly bacteria that harbor a transferable antibiotic resistance gene, complicates the management of bacterial infections. In this study, we investigated the presence of the transferable colistin resistance mcr genes in patients with leukemia in Spain. METHODS: 217 fecal samples collected in 2013-2015 from 56 patients with acute leukemia and colonized with MDR Enterobacteriaceae strains, were screened on September 2017 for the presence of the colistin resistance mcr genes (mcr-1 to -5) by multiplex PCR. mcr positive strains selected on LBJMR and MacConkey supplemented with colistin (2 µg/ml) media were phenotypically and molecularly characterized by antimicrobial susceptibility testing, minimum inhibitory concentration, multilocus sequence typing and plasmid characterization. RESULTS: Among 217 fecal samples, 5 samples collected from 3 patients were positive for the presence of the mcr-1 colistin-resistance gene. Four Escherichia coli strains were isolated and exhibited resistance to colistin with MIC = 4 µg/ml. Other genes conferring the resistance to ß-lactam antibiotics have also been identified in mcr-1 positive strains, including blaTEM-206 and blaTEM-98. Three different sequence types were identified, including ST1196, ST140 and ST10. Plasmid characterization allowed us to detect the mcr-1 colistin resistance gene on conjugative IncP plasmid type. CONCLUSION: To the best of our knowledge, we have identified the mcr-1 gene for the first time in leukemia patients in Spain. In light of these results, strict measures have been implemented to prevent its dissemination.

Mycobacterium ulcerans Experimental Dormancy.

Whether Mycobacterium ulcerans, the etiological agent of Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, has not been established. Six M. ulcerans strains were exposed to a progressive depletion in oxygen for 2 months, using the Wayne model of dormancy previously described for M. tuberculosis, and further examined by microscopy after staining of dynamic, dormant, and dead mycobacteria (DDD staining), microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls. Mycobacterium ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile red-stained intracellular lipid droplets and a 14- to 20-day regrowth when exposed to ambient air, consistent with dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD staining and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, warrants further investigations in the natural ecological niches where M. ulcerans thrive as well as in Buruli ulcer lesions.

Genetic Diversity and Virulence Profile of Methicillin and Inducible Clindamycin-Resistant Staphylococcus aureus Isolates in Western Algeria.

Staphylococcus aureus causes a wide range of life-threatening infections. In this study, we determined its prevalence in the hospital environment and investigated nasal carriage among healthcare workers and patients admitted to a hospital in western Algeria. A total of 550 specimens were collected. An antibiogram was performed and the genes encoding resistance to methicillin, inducible clindamycin and toxins were sought among the 92 S. aureus isolates. The spread of clones with a methicillin- and/or clindamycin-resistance phenotype between these ecosystems was studied using genomic analysis. A prevalence of 27%, 30% and 13% of S. aureus (including 2.7%, 5% and 1.25% of MRSA) in patients, healthcare workers and the hospital environment were observed, respectively. The presence of the mecA, erm, pvl and tsst-1 genes was detected in 10.9%, 17.4%, 7.6% and 18.5% of samples, respectively. Sequencing allowed us to identify seven sequence types, including three MRSA-IV-ST6, two MRSA-IV-ST80-PVL+, two MRSA-IV-ST22-TSST-1, two MRSA-V-ST5, and one MRSA-IV-ST398, as well as many virulence genes. Here, we reported that both the hospital environment and nasal carriage may be reservoirs contributing to the spread of the same pathogenic clone persisting over time. The circulation of different pathogenic clones of MRSA, MSSA, and iMLSB, as well as the emergence of at-risk ST398 clones should be monitored.

Lactobacillus supports Clostridiales to restrict gut colonization by multidrug-resistant Enterobacteriaceae.

Infections by multidrug-resistant Enterobacteriaceae (MRE) are life-threatening to patients. The intestinal microbiome protects against MRE colonization, but antibiotics cause collateral damage to commensals and open the way to colonization and subsequent infection. Despite the significance of this problem, the specific commensals and mechanisms that restrict MRE colonization remain largely unknown. Here, by performing a multi-omic prospective study of hospitalized patients combined with mice experiments, we find that Lactobacillus is key, though not sufficient, to restrict MRE gut colonization. Lactobacillus rhamnosus and murinus increase the levels of Clostridiales bacteria, which induces a hostile environment for MRE growth through increased butyrate levels and reduced nutrient sources. This mechanism of colonization resistance, an interaction between Lactobacillus spp. and Clostridiales involving cooperation between microbiota members, is conserved in mice and patients. These results stress the importance of exploiting microbiome interactions for developing effective probiotics that prevent infections in hospitalized patients.

First Clinical Cases of KPC-2-Producing Klebsiella pneumoniae ST258 in Algeria and Outbreak of Klebsiella pneumoniae ST101 Harboring blaOXA-48 Gene in the Urology Department of Annaba Hospital.

Objectives: The aim of this study was to characterize the molecular mechanisms of carbapenem resistance in Klebsiella pneumoniae isolated from the urology department of Annaba hospital, Algeria. Methods: Between January 2015 and September 2017, 14 carbapenem-resistant K. pneumoniae strains were isolated during routine surveillance work at Ibn Roched hospital of Annaba, Algeria, from the urology department. Theses strains were recovered, and carbapenem resistance mechanisms were investigated. The strains were identified by using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Antibiotic susceptibility was assessed by using the Kirby-Bauer method, whereas minimum inhibitory concentration of imipenem/ertapenem and colistin was determined by Etest and broth microdilution methods, respectively. Carbapenem resistance determinants were studied by using PCR and sequencing methods and analyzed by BLAST against the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) database. Clonal relationship of strains was performed by using multilocus sequence typing (MLST). Transferability of carbapenem resistance genes was assessed by conjugation and transformation experiments. Results: Fourteen carbapenem-resistant K. pneumoniae isolates were found to be resistant to the eight ß-lactam antibiotics tested (except to imipenem for two isolates). Carbapenemase production was positive for all isolates. Molecular characterization revealed that blaKPC-2 and blaOXA-48 genes were detected in 3 (21.4%) and 11 isolates (78.6%), respectively. Other ß-lactamases genes were identified, including blaCTX-M-15, blaSHV-1-or 12, and blaTEM-1. MLST revealed that the 14 isolates belonged to 2 different sequence types (STs), including ST101 (11 OXA-48-producing K. pneumoniae) and ST258 (3 KPC-2-producing K. pneumoniae). PCR amplifications for blaKPC-2 and blaOXA-48 carbapenemases genes performed on extracted plasmids, showed positive results, suggesting that both carbapenemase genes were probably borne by plasmids. Conclusion: We report here the first identification of KPC-2-producing K. pneumoniae ST258 in Algerian hospitals and an outbreak of OXA-48-producing K. pneumoniae isolates ST101 in the urology department of Ibn Roched hospital located in Annaba, Algeria.

Infections Due to Carbapenem-Resistant Bacteria in Patients With Hematologic Malignancies.

In developed countries, hematological malignancies (HM) account for 8 to 10% of cancers diagnosed annually and one-third of patients with HM (HMP) are expected to die from their disease. The former wide spectrum "magic bullet," imipenem, has been ousted by the emergence of carbapenem resistant (CR) pathogens. In endemic areas, infections with CR-bacteria occur in vulnerable patients, notably in HMP, who suffer from high mortality related to infectious complications. In this work, we reviewed epidemiologic and clinical factors associated with CR-infections in adult HMP and data on CR-related mortality and antibiotic treatments in this population. We found that resistance profile of strains involved in HMP infections, mainly bacteremia, reflect local epidemiology. Significant risk factors for infections with CR-bacteria include sex male, age around 50 years old, acute leukemia, selvage chemotherapy, neutropenia, and digestive colonization by CR-bacteria. Mortality rate is high in HMP infected with CR-Enterobacteriaceae, more particularly in case of acute myeloid leukemia and unresolved neutropenia, due to inappropriate empiric management and delayed administration of targeted antibiotics, such as tigecycline, colistin, or new associations of active drugs. Thus, we developed an algorithm for clinicians, assessing the incremental risk for CR-bacterial infection occurrence and mortality in febrile HMP, to guide decisions related to empirical therapeutic strategies.

Spread of Carbapenem and Colistin-Resistant Klebsiella pneumoniae ST512 Clinical Isolates in Israel: A Cause for Vigilance.

OBJECTIVES: Genomic and phenotypic characterization of resistance mechanisms to carbapenems and colistin in Klebsiella pneumoniae strains isolated from the Shaare Zedek Medical Center (Jerusalem, Israel). RESULTS: The 15 K. pneumoniae isolates studied present a high level of resistance to the antibiotics tested. Microbiological tests revealed production of carbapenemase enzymes by all isolates. ABI SOLiD sequencing of K. pneumoniae genomes generated between 5,033,665 and 8,876,861 million reads per genome. The genomic study revealed that carbapenem resistance was mediated by production of the KPC-3 enzyme in 13 isolates and NDM-1 enzyme in the remaining 2 isolates. In addition, colistin resistance was induced either by a missense mutation in the mgrB gene or inactivation of mgrB by an IS5-like insertion sequence. The mobile genetic element, transposon Tn4401, was identified in all genomes harboring the blaKPC gene. The 15 K. pneumoniae strains were assigned to 4 different sequence types, including ST16, ST76, ST258, and ST512. CONCLUSION: In this study, we report the usefulness of whole-genome sequencing in detection of antibiotic resistance mechanisms and in highlighting the emergence of the carbapenem and colistin-resistant K. pneumoniae clone ST512 in Israeli hospitals.

Genomic characterization of Citrobacter freundii strains coproducing OXA-48 and VIM-1 carbapenemase enzymes isolated in leukemic patient in Spain.

Background: The emergence of carbapenemase-producing (CP) Citrobacter freundii poses a significant threat to public health, especially in high-risk populations. In this study, whole genome sequencing was used to characterize the carbapenem resistance mechanism of three C. freundii clinical isolates recovered from fecal samples of patients with acute leukemia (AL) from Spain. Materials and methods: Twelve fecal samples, collected between 2013 and 2015 from 9 patients with AL, were screened for the presence of CP strains by selecting them on MacConkey agar supplemented with ertapenem (0.5 mg/L). Bacteria were identified by MALDI-TOF mass spectrometry and were phenotypically characterized. Whole genome sequencing of C. freundii isolates was performed using the MinION and MiSeq Illumina sequencers. Bioinformatic analysis was performed in order to identify the molecular support of carbapenem resistance and to study the genetic environment of carbapenem resistance encoding genes. Results: Three carbapenem-resistant C. freundii strains (imipenem MIC≥32 mg/L) corresponding to three different AL patients were isolated. Positive modified Carba NP test results suggested carbapenemase production. The genomes of each C. freundii tested were assembled into a single chromosomal contig and plasmids contig. In all the strains, the carbapenem resistance was due to the coproduction of OXA-48 and VIM-1 enzymes encoded by genes located on chromosome and on an IncHI2 plasmid, respectively. According to the MLST and the SNPs analysis, all strains belonged to the same clone ST169. Conclusion: We report in our study, the intestinal carrying of C. freundii clone ST169 coproducing OXA-48 and VIM-1 identified in leukemic patients.

Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Clinical_Isolates: Algeria.

This study investigates the molecular mechanisms of colistin and carbapenem resistance in Klebsiella pneumoniae ST101 strains. The three K. pneumoniae carried blaCTX-M-15, blaTEM-183, and blaSHV-106 genes and two coharbored blaOXA-48. As for colistin resistance, the isolates had amino acid substitutions in PmrA/B and a truncated mgrB gene in one isolate.

Evaluation of different testing tools for the identification of non-gonococcal Neisseria spp. isolated from Lebanese male semen: a strong and significant association with infertility.

PURPOSE: The aim was to evaluate several microbiological tools for the identification of non-gonococcal Neisseria spp. isolated from semen samples from Lebanese men and to determine the putative link between the presence of Neisseria commensal species and infertility. METHODOLOGY: Within a cross-sectional retrospective study design, the whole population included in this investigation was divided in 2 categories: 173 patients with symptoms of infertility and 139 patients with normal seminograms. Epidemiological and microbiological investigations were performed for 59 strains of Neisseria through several phenotypic and genotypic tools, including seminograms, an analytical profile index of Neisseria and Haemophilus (API-NH), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), porA PCR, 16S rRNA and rplF gene sequencing, and antimicrobial susceptibility testing. RESULTS: The risk of Neisseria infection was twice as high in infertile patients compared to the control group [odds ratio (OR): 1.95, confidence interval (CI): 1.05-3.65, P =0.03]. Unreliable diagnosis of Neisseria urogenital infection has serious health and social consequences. Our findings showed that API-NH and 16S rRNA sequencing are poor tools to identify Neisseria at the species level. Therefore, reliable diagnosis of cases using MALDI-TOF MS and/or rplF sequencing is needed to provide critical treatment decisions and prevent antimicrobial resistance spreading in the community. CONCLUSION: This work predicted a strong and significant association between the presence of Neisseria spp. in semen and male infertility among the Lebanese population. For a better understanding of this association, it is recommended that more genomic and large-scale epidemiological investigations are undertaken to reach definitive conclusions.

Occurrence of Carbapenemase-Producing Enterobacteriaceae Isolates in the Wildlife: First Report of OXA-48 in Wild Boars in Algeria.

The aim of the present study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) isolates from wild boars and Barbary macaques in Algeria. Fecal samples were collected from wild boars (n = 168) and Barbary macaques (n = 212), in Bejaia, Algeria, between September 2014 and April 2016. The isolates were identified and antimicrobial susceptibility was determined. Carbapenem resistance determinants were studied using PCR and sequencing, while clonal relatedness was performed using multilocus sequence typing (MLST). PCR was used to investigate certain virulence genes. Three CPE isolates from three different samples (1.8%) recovered from wild boars were identified as Escherichia coli (two isolates) and Klebsiella pneumoniae (one isolate). These isolates were resistant to amoxicillin, amoxicillin-clavulanate, tobramycin, ertapenem, and meropenem. The results of PCR and sequencing analysis showed that all three isolates produced the OXA-48 enzyme. The MLST showed that the two E. coli isolates were assigned to the same sequence type, ST635, and belonged to phylogroup A, whereas K. pneumoniae strain belonged to ST13. The K. pneumoniae strain was positive for multiple virulence factors, whereas no virulence determinants were found in E. coli isolates. This is the first report of OXA-48-producing Enterobacteriaceae in wild animals from Algeria and Africa.

First Report of the Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli ST405 Isolated from Wildlife in Bejaia, Algeria.

BACKGROUND: The aim of the present study was to screen for the presence of mcr-1 gene in Enterobacteriaceae isolated from Barbary macaques (Macaca sylvanus) in Algeria. MATERIALS AND METHODS: From January to April 2016, a total of 86 fresh stool samples from Barbary macaques were collected in the Toudja forest (Bejaia, Algeria). After isolation, the isolates were identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Antibiotic susceptibility was determined by disk diffusion method, and minimum inhibitory concentration (MIC) of colistin was determined by E-test. Polymerase chain reaction (PCR) and sequencing were used to identify mcr-1gene. The sequence type (ST) was done using multilocus sequence typing. Phylogenetic groups of Escherichia coli and the presence of virulence genes were determined by PCR. Transfer of mcr-1 and extended-spectrum ß-lactamase genes was assessed by conjugation and transformation experiments. RESULTS: E. coli M076 isolate was found resistant to colistin with a MIC of 4 mg/L and to other antibiotic families, including ß-lactams, aminoglycosides, and fluoroquinolones. PCR and sequencing revealed that this isolate harbored the mcr-1, blaCTX-M-15, blaTEM-1, and qnrB19 genes. This isolate was assigned to ST405, belonged to phylogroup D, and contains only one virulence gene fyuA (siderophore uptake receptor). Neither transconjugants nor transformants were obtained for this isolate. CONCLUSION: In this study, we report the detection of mcr-1 gene producing E. coli from wild animals.

Molecular characterization of carbapenem-resistant Gram-negative bacilli clinical isolates in Algeria.

OBJECTIVES: The aims of this study were to investigate the occurrence of carbapenem-resistant Gram-negative bacilli (GNB) isolated from inpatients and outpatients in Algeria between July and September 2015, and to screen their resistance mechanisms and genetic relatedness. MATERIALS AND METHODS: A total of 68 non-redundant isolates were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Carbapenemase activity was carried out using modified Carba NP test, EDTA assay, and the modified Hodge test. Molecular characterization of carbapenemases and extended-spectrum ß-lactamase (ESBL) genes were detected by standard PCR and sequencing. Genotyping of carbapenem-resistant isolates was performed by multilocus sequence typing (MLST) analysis. RESULTS: Of the 68 GNB isolates, 13 (19%) showed reduced susceptibility to carbapenems, including, four Klebsiella pneumoniae, one Escherichia coli, six Acinetobacter baumannii, and two Pseudomonas aeruginosa. blaOXA-48 gene was detected in the five Enterobacteriaceae isolates, and blaOXA-23 was identified in all A. baumannii isolates. OprD mutations were revealed in the two P. aeruginosa isolates. A total of 11 out of the 13 carbapenem-resistant GNB were detected in inpatients, and the two remaining strains were isolated from outpatients. Molecular typing showed the presence of four sequence types (STs) among the OXA-48-producing K. pneumoniae isolates: ST101, ST147, ST163, and ST2017. ST533 was identified for the OXA-48 producing E. coli isolate. All of the A. baumannii and P. aeruginosa were assigned to the international clonal lineages ST2 and ST654, respectively. CONCLUSION: This study reports the first detection of the epidemic multidrug-resistant lineage, K. pneumoniae ST147 coproduced blaOXA-48 and ESBL genes in Algeria and represents the first description of OXA-48-producing E. coli ST533 and K. pneumoniae ST163 and ST2017. In addition, this study describes for the first time the emergence of OXA-48-producing E. coli and K. pneumoniae in the community in Algeria, leading to major problems for managing microbial infections.