Thongpan Leangapichart

Real-time quantitative PCR assay with Taqman® probe for rapid detection of MCR-1 plasmid-mediated colistin resistance

Here we report the development of two rapid real-time quantitative PCR assays with TaqMan® probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli) with a calibration curve that was linear from 101 to 108 DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing.

Acquisition of mcr-1 Plasmid-Mediated Colistin Resistance in Escherichia coli and Klebsiella pneumoniae during Hajj 2013 and 2014

Aplasmid-mediated transferable colistin resistance gene, mcr-1, was recently described in China (1) and was rapidly reported in several other countries (2). The spread of the mcr-1 gene was not only reported in Escherichia coli but also associated with other Enterobacteriaceae species isolated from human clinical samples, farm animals, and travelers (2, 3). However, whether or not the mcr-1 gene is acquired during the Hajj (the Muslim pilgrimage to Mecca) remains unknown. We conducted two cohort studies of pilgrims traveling to Mecca in 2013 (22 September to 23 October) (4, 5) and in 2014 (19 September to 12 October) (6). A total of 440 rectal swab samples were collected from pilgrims (in 2013, 129 pilgrims [before and after their pilgrimage]; in 2014, 92 pilgrims before and 90 pilgrims after their pilgrimage) and were then screened for the presence of the mcr-1 gene by real-time PCR and results confirmed by standard PCR and sequencing as described previously (7). All PCR-positive samples were then tested in an attempt to isolate mcr-1-resistant strains by culture on Cepacia agar (bioMérieux, Marcy-l’Étoile, France). Different colonies were tested by matrixassisted laser desorption ionization–time of flight (MALDI-TOF), antibiotic susceptibility testing (EUCAST), Etest (MIC susceptibility, 2 mg/liter), PCR, sequencing of the mcr-1 and extendedspectrum-beta-lactamase (ESBL) genes (blaCTX-M, blaTEM, and blaSHV) (5), and multilocus sequence typing (MLST) analysis (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/ and http://bigsdb.web .pasteur.fr/klebsiella/klebsiella.html). All mcr-1 and ESBL gene sequencing results were then analyzed with NCBI database. The prevalences of mcr-1-positive isolates determined by PCR in rectal swabs of pilgrims were similar in 2013 and 2014, and the prevalence was significantly higher upon return (in 2013, 1.55% [2/129] before the pilgrimage versus 8.53% [11/129] after the pilgrimage [P 0.0104]; in 2014, 1.02% [1/92] before the pilgrimage versus 9.18% [9/90] after the pilgrimage [P 0.0091]). Ten E. coli isolates and 1 K. pneumoniae isolate from 23 pilgrims who were mcr-1 positive by PCR were successfully identified by culture (Table 1). The sequence of the detected mcr-1 gene showed 100% identity with the published sequence (1). Our colistin-resistant isolates were not resistant to all antibiotics (Table 1). MICs of colistin ranged from 3 to 4 mg/liter. Two unrelated pilgrims (no. 95 and 96) carried the common sequence type of E. coli, ST10, likely suggesting that the isolates from those two pilgrims represented the same clone. Also, two other unrelated pilgrims (no. 6 and 117) carried the same sequence type of E. coli, ST648. Conversely, two Moroccan pilgrims (no. 134 and 143) who formed a

Acquisition of a High Diversity of Bacteria during the Hajj Pilgrimage, Including Acinetobacter baumannii with blaOXA-72 and Escherichia coli with blaNDM-5 Carbapenemase Genes

ABSTRACT Pilgrims returning from the Hajj (pilgrimage to Mecca) can be carriers of multidrug-resistant bacteria (MDR). Pharyngeal and rectal swab samples were collected from 98 pilgrims before and after they traveled to the Hajj in 2014 to investigate the acquisition of MDR bacteria. The bacterial diversity in pharyngeal swab samples was assessed by culture with selective media. There was a significantly higher diversity of bacteria in samples collected after the return from the Hajj than in those collected before (P = 0.0008). Surprisingly, Acinetobacter baumannii strains were isolated from 16 pharyngeal swab samples (1 sample taken during the Hajj and 15 samples taken upon return) and 26 post-Hajj rectal swab samples, while none were isolated from samples taken before the Hajj. Testing of all samples by real-time PCR targeting blaOXA-51 gave positive results for only 1% of samples taken during the Hajj, 21/90 (23.3%) pharyngeal swab samples taken post-Hajj, and 35/90 (38.9%) rectal swab samples taken post-Hajj. One strain of A. baumannii isolated from the pharynx was resistant to imipenem and harbored a blaOXA-72 carbapenemase gene. Multilocus sequence typing analysis of 43 A. baumannii isolates revealed a huge diversity of 35 sequence types (STs), among which 18 were novel STs reported for the first time in this study. Moreover, we also found one Escherichia coli isolate, collected from a rectal swab sample from a pilgrim taken after the Hajj, which harbored blaNDM-5, blaCTX-M-15, blaTEM-1, and aadA2 (ST2659 and ST181). In conclusion, pilgrims are at a potential risk of acquiring and transmitting MDR Acinetobacter spp. and carbapenemase-producing Gram-negative bacteria during the Hajj season.

Plasmid-Mediated mcr-1 Gene in Colistin-Resistant Clinical Isolates of Klebsiella pneumoniae in France and Laos

Recently, Yi-Yun Liu et al. reported the emergence of plasmid-mediated colistin resistance involving the mcr-1 gene from Escherichia coli and Klebsiella pneumoniae isolates from animals, food, and humans in China ([1][1]). Because of the plasmidic location of this new gene, which encodes a

Acquisition of Extended-Spectrum β-Lactamases by Escherichia coli and Klebsiella pneumoniae in Gut Microbiota of Pilgrims during the Hajj Pilgrimage of 2013

ABSTRACT We reported the acquisition of extended-spectrum-β-lactamase (ESBL)-producing bacteria in rectal samples of 129 pilgrims during the 2013 Hajj (pilgrimage to Makkah). When returning from the Hajj, there was a significant increase in the number of pilgrims carrying E. coli resistant to ceftriaxone (P = 0.008). The CTX-M gene was detected in rectal samples, with the detection rate increasing from 10.08% to 32.56% of samples after the Hajj (P < 0.001).

Whole-Genome Sequence of a blaOXA-48-Harboring Raoultella ornithinolytica Clinical Isolate from Lebanon

ABSTRACT We analyzed the whole-genome sequence of a blaOXA-48-harboring Raoultella ornithinolytica clinical isolate from a patient in Lebanon. The size of the Raoultella ornithinolytica CMUL058 genome was 5,622,862 bp, with a G+C content of 55.7%. We deciphered all the molecular mechanisms of antibiotic resistance, and we compared our genome to other available R. ornithinolytica genomes in GenBank. The resistome consisted of 9 antibiotic resistance genes, including a plasmidic blaOXA-48 gene whose genetic organization is also described.

Deciphering Heteroresistance to Colistin in a Klebsiella pneumoniae Isolate from Marseille, France

ABSTRACT Here, we report the description of a colistin-heteroresistant Klebsiella pneumoniae isolate fortuitously isolated from the stool sample of a patient with suspicion of tuberculosis in a public hospital of Marseille, France. In the colistin-resistant subpopulation, a mutation in the mgrB gene leading to a premature stop codon was found, and the hypermucoviscous phenotype was lost. Susceptibility to other antibiotics remained unchanged. To our knowledge, this is the first identification of such a colistin-heteroresistant Klebsiella pneumoniae isolate in France.

Characterization of bla OXA-538 , a new variant of bla OXA-48 in Shewanella xiamenensis isolated from River water in Algeria

OBJECTIVES In this study, the presence of carbapenemase genes in Shewanella xiamenensis strains isolated from river water in Béjaïa, Algeria, was investigated. METHODS Four isolates of S. xiamenensis were isolated from water from Soummam River. The isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and sequencing of the 16S rRNA and gyrB genes. Isolates were subjected to antimicrobial susceptibility testing. Carbapenemase production was screened using phenotypic tests. PCR and sequencing were used to identify carbapenemase genes in the isolates. The genetic context of the blaOXA-48-like gene was investigated by sequencing the whole genome of strain AS58. RESULTS All four S. xiamenensis strains harboured blaOXA-48-like genes. They exhibited different resistance patterns and had imipenem minimum inhibitory concentrations (MICs) of ≥0.5mg/L. Sequencing of blaOXA-48-like genes from the S. xiamenensis isolates showed that two strains harboured blaOXA-181, one strain harboured blaOXA-199 and one strain exhibited a new variant of the blaOXA-48-like gene, named blaOXA-538. This new variant shared 98% nucleotide identity with blaOXA-162, with three amino acid changes (G201A, A213G and I219F). Conjugation assays with Escherichia coli J53 recipient were performed but no transconjugants were obtained. Analysis of the genome of AS58 Touati strain confirmed the chromosomal location of the blaOXA-538 gene. CONCLUSION This study showed that environmental water holds a diversity of S. xiamenensis strains harbouring blaOXA-48-like genes and may play an important role in the dissemination and spread of these genes from the environment to humans.

Genome sequence of “Leucobacter massiliensis” sp. nov. isolated from human pharynx after travel to the 2014 Hajj

“Leucobacter massiliensis” strain 122RC15T sp. nov. is a new species within the genus Leucobacter. The genome of this strain is described here. It was isolated from the pharynx of a 76-year-old Algerian female after travelling from the 2014 Hajj. “Leucobacter massiliensis” is a Gram-positive, aerobic bacillus. Here we describe the features including complete genome and annotation of this strain. The 3 136 406-bp long genome contains 2797 protein-coding genes and 49 RNA genes.