Screening of colistin-resistant bacteria in livestock animals from France.

Colistin is frequently used as a growth factor or treatment against infectious bacterial diseases in animals. The Veterinary Division of the European Medicines Agency (EMA) restricted colistin use as a second-line treatment to reduce colistin resistance. In 2020, 282 faecal samples were collected from chickens, cattle, sheep, goats, and pigs in the south of France. In order to track the emergence of mobilized colistin resistant (mcr) genes in pigs, 111 samples were re-collected in 2021 and included pig faeces, food, and water from the same location. All samples were cultured in a selective Lucie Bardet Jean-Marc Rolain (LBJMR) medium and colonies were identified using MALDI-TOF mass spectrometry and then antibiotic susceptibility tests were performed. PCR and Sanger sequencing were performed to screen for the presence of mcr genes. The selective culture revealed the presence of 397 bacteria corresponding to 35 different bacterial species including Gram-negative and Gram-positive. Pigs had the highest prevalence of colistin-resistant bacteria with an abundance of intrinsically colistin-resistant bacteria and from these samples one strain harbouring both mcr-1 and mcr-3 has been isolated. The second collection allowed us to identify 304 bacteria and revealed the spread of mcr-1 and mcr-3 in pigs. In the other samples, naturally, colistin-resistant bacteria were more frequent, nevertheless the mcr-1 variant was the most abundant gene found in chicken, sheep, and goat samples and one cattle sample was positive for the mcr-3 gene. Animals are potential reservoir of colistin-resistant bacteria which varies from one animal to another. Interventions and alternative options are required to reduce the emergence of colistin resistance and to avoid zoonotic transmissions.

clbP Gene, a Potential New Member of the ß-Lactamase Family.

The colibactin island (pks) of Escherichia coli formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The clbP, an unusual essential gene, is found in the operon structure with the clbS gene in the pks-encoded machinery. Interestingly, the clbP gene has been annotated as a ß-lactamase but no previous study has reported its ß-lactamase characteristics. In this study, we (i) investigated the ß-lactamase properties of the clbP gene in silico by analysing its phylogenetic relationship with bacterial ß-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial ß-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including ß-lactamase, nuclease, and ribonuclease using protein expression and purification from an E. coli BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the clbP gene within the pks-island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of ß-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its ß-lactamase activity, likely due to the fact that the clbP gene requires co-expression with other genes, such as the genes present in the pks-island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity.

fosM, a New Family of Fosfomycin Resistance Genes Identified in Bacterial Species Isolated from Human Microbiota.

Fosfomycin is a decades-old antibiotic, currently reused because of its activity against multidrug-resistant bacteria. Here, we used a combined in vitro/in silico approach to search for fosfomycin resistance determinants in 25 new bacterial species isolated from the human microbiota. Putative resistance genes were cloned into a susceptible Escherichia coli strain. MIC values increased from 1 µg/ml to 1,024 µg/ml. Here, we report a new family of potential chromosomal fosfomycin resistance genes, named fosM.

Bhargavaea massiliensis sp. nov. and Dietzia massiliensis sp. nov., Novel Bacteria Species Isolated from Human Urine Samples in Nigeria.

Two novel bacteria species designated Marseille-Q1000T and Marseille-Q0999T were isolated from urine samples of patients in Sokoto, Northwest-Nigeria. They were Gram-positive bacteria and belong to two different genera, Bhargavaea and Dietzia. The genome size and G + C content of Marseille-Q1000T and Marseille-Q0999T were 3.07 and 3.51 Mbp with 53.8 and 71.0 mol% G + C content, respectively. The strains exhibited unique phenotypic and genomic features that are substantially different from previously known bacterial species with standing in nomenclature. On the basis of the phenotypic, phylogenetic and genomic characteristics, strains Marseille-Q0999T (= CSURQ0999 = DSM 112394) and Marseille-Q1000T (= CSURQ1000 = DSM 112384) were proposed as the type strains of Bhargavaea massiliensis sp. nov., and Dietzia massiliensis sp. nov., respectively.

First Isolation and Clinical Case of Brevundimonasdiminuta in a Newborn with Low Birth Weight, in Democratic Republic of Congo: A Case Report.

Brevundimonas diminuta is rarely described in clinical specimens, never at the umbilical stump. Most of the reported cases are in patients with underlying pathologies. We must integrate this microorganism in the etiological agents of nosocomial infections, but much remains to be understood about its virulence. We present a case of umbilical stump infection (omphalitis) caused by B. diminuta, in a preterm and hypotrophic new-born and discuss the diagnosis of this bacterium and its role as responsible of nosocomial neonatal infections.

Inactivation of thymidine kinase as a cause of resistance to zidovudine in clinical isolates of Escherichia coli: a phenotypic and genomic study.

OBJECTIVES: The antiviral zidovudine has been recently identified as an active drug against resistant Enterobacteriaceae, but prevalence of resistance to this compound remains unknown. The aim was to estimate the prevalence of clinical Escherichia coli isolates resistant to zidovudine and to decipher the mechanism of zidovudine resistance. METHODS: We screened 537 isolates on zidovudine-containing agar plates and studied their thymidine kinase (tdk) gene sequences, the putative target involved in zidovudine resistance. Moreover, sequence analysis of 633 complete genomes of E. coli was performed to investigate mutation in the tdk gene. A comparative genomic analysis was done on an in vitro zidovudine-resistant mutant. RESULTS: After screening on our medium containing 2.7 mg/L (10 µM) zidovudine, nine strains had a zidovudine MIC >26.7 mg/L. The gene was absent in three isolates, inactivated by an IS (IS1X2 and ISApl1) in two isolates and mutated in four isolates. A genomic analysis of 633 E. coli genomes showed heterogeneity of the tdk gene sequence, with 27 different sequences. Among them, three genomes showed an inactivation of the gene (IS, stop codon and no tdk gene sequence). The in vitro mutant E. coli had 27 SNPs in eight genes of the core genome compared with the initial strain. CONCLUSIONS: Our study reports zidovudine-resistant clinical isolates of E. coli, presumably related to tdk inactivation. Diversity of Tdk in bacterial genomes can be large. Other mechanisms need to be considered in zidovudine resistance. The use of zidovudine in antibiotic-resistant infections needs to be in combination and should be tested before clinical administration.

Promiscuous Enzyme Activity as a Driver of Allo and Iso Convergent Evolution, Lessons from the ß-Lactamases.

The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. The more the evolution of a character is probable, the more the convergent evolution will be witnessed, and consequently, convergent evolution could mean that the convergent character evolution results as a combination of these two factors. We investigated this phenomenon by studying the convergent evolution of biochemical functions. For the investigation we used the case of ß-lactamases. ß-lactamases hydrolyze ß-lactams, which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. ß-lactamase activity is present in two different superfamilies: the metallo-ß-lactamase and the serine ß-lactamase. The mechanism used to hydrolyze the ß-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further showed that the ß-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explained by the promiscuous ß-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part by positive selection, as the organisms having ß-lactamase activity allows them to resist organisms that secrete ß-lactams. Indeed, an organism that has a mutation that increases the ß-lactamase activity will be selected, as the organisms having this activity will have an advantage over the others.

An Integrative Database of ß-Lactamase Enzymes: Sequences, Structures, Functions, and Phylogenetic Trees.

ß-Lactamase enzymes have attracted substential medical attention from researchers and clinicians because of their clinical, ecological, and evolutionary interest. Here, we present a comprehensive online database of ß-lactamase enzymes. The current database is manually curated and incorporates the primary amino acid sequences, closest structural information in an external structure database (the Protein Data Bank [PDB]) and the functional profiles and phylogenetic trees of the four molecular classes (A, B, C, and D) of ß-lactamases. The functional profiles are presented according to the MICs and kinetic parameters that make them more useful for the investigators. Here, a total of 1,147 ß-lactam resistance genes are analyzed and described in the database. The database is implemented in MySQL and the related website is developed with Zend Framework 2 on an Apache server, supporting all major web browsers. Users can easily retrieve and visualize biologically important information using a set of efficient queries from a graphical interface. This database is freely accessible at http://ifr48.timone.univ-mrs.fr/beta-lactamase/public/.

Investigation of multidrug-resistant ST2 Acinetobacter baumannii isolated from Saint George hospital in Lebanon.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen causing various nosocomial infections. The spread of multidrug-resistant A. baumannii is a major public health problem. The aim of this study was to investigate the molecular epidemiology and the genetic support of multidrug-resistant A. baumannii isolates collected from Saint-Georges Hospital in Lebanon. METHODS: Between January and August 2016, 31 A. baumannii isolates were collected from sputum samples of patients infected with ventilator-associated pneumonia (VAP) and treated with colistin-carbapenem combination therapy. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemases, extended spectrum ß-lactamases encoding genes and mcr-1/2 genes were investigated by RT-PCR and standard PCR. The epidemiological relatedness of the strains was studied using MLST analysis. RESULTS: Most of the isolates exhibited multidrug-resistant phenotypes. All the isolates were carbapenem-resistant and among them, 30 carried the class D carbapenemase blaoxa-23 gene while one isolate carried blaoxa-72 gene. MLST results revealed three sequence types, namely ST2, ST699, and ST627. Isolates having ST2 were the most prevalent clone (29/31, 93.5%). CONCLUSIONS: This study shows a nosocomial spread of multidrug-resistant A. baumannii ST2 having blaOXA-23 gene in Saint-George in Lebanon. Monitoring and control measures need to be adopted to avoid the spread of A. baumannii to patients.

Colistin Heteroresistance and Involvement of the PmrAB Regulatory System in Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem, and colistin is increasingly being used as a last-resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance (HR) to colistin have been described. The purpose of the present study was to investigate the role of the PmrAB regulatory pathway in laboratory-selected mutants representative of global epidemic strains. From three unrelated A. baumannii clinical strains (sequence types 2, 3, and 20), eight colistin-resistant mutants were selected. Half of the mutants showed HR to colistin according to the reference method (population analysis profiling), whereas the other half exhibited stable resistance. M12I mutation within pmrA and M308R, S144KLAGS, and P170L mutations for pmrB were associated with HR to colistin, while T235I, A226T, and P233S mutations within pmrB were associated with stable resistance. The transcript levels of the pmrCAB operon were upregulated in all the mutants. Compensatory mutations were explored for some mutants. A single mutant (T235I mutant) displayed a compensatory mutation through ISAba1 mobilization within the pmrB gene that was associated with the loss of colistin resistance. The mutant resistance phenotype associated with T235I was partially restored in a trans-complementation assay turning to HR. The level of colistin resistance was correlated with the level of expression of pmrC in the trans-complemented strains. This report shows the role of different mutations in the PmrAB regulatory pathway and warns of the development of colistin HR that could be present but not easily detected through routine testing.

Characterization of Staphylococcus aureus Isolated from Food Products in Western Algeria.

The current study aimed to characterize Staphylococcus aureus isolates from foodstuffs collected from western Algeria. A total of 153 S. aureus isolates from various raw and processed foods were obtained and identified using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Isolates were characterized by antimicrobial susceptibility testing and toxin gene detection. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were identified by detection of the mecA gene and characterized by staphylococcal cassette chromosome mec (SCCmec) typing. We found that 30.9% (153/495) of food samples were contaminated with S. aureus. Thirty-three (21.5%) S. aureus isolates were identified as MRSA, and 16.9% (26/153) carried the mecA gene. Three SCCmec types were identified of which type IV was the most common (69.2%) followed by type V (15.3%) and type II (7.6%). Two MRSA isolates were not typable with SCCmec typing. None of the examined isolates harbored mecC. Furthermore, 14.3% (22/153) of the isolates were toxigenic S. aureus. The cytotoxin gene pvl was detected in 11.1% of the S. aureus isolates. This gene was more commonly detected (76.4%) in MRSA isolates than in methicillin-suceptible Staphylococcus aureus (MSSA) isolates. The tsst-1 gene coding for toxic shock syndrome toxin was isolated rarely (3.2%) and only in MSSA isolates. According to disk diffusion test results, 70 isolates were resistant to only one antimicrobial drug, and 51 (33.3%) isolates were multidrug resistant. Other 32 isolates were susceptible to all antibiotics. Our study highlights, for the first time, a high prevalence of multidrug-resistant S. aureus isolates carrying pvl or tsst-1 found in food products in Algeria. The risk of MRSA transmission through the food chain cannot be disregarded, particularly in uncooked foods.

Prophages and adaptation of Staphylococcus aureus ST398 to the human clinic.

BACKGROUND: It has been suggested that prophages in the ST398 S. aureus clone are responsible for expanding ST398's spectrum of action and increasing its ability to cause human infections. We carried out the first characterization of the various prophages carried by 76 ST398 bloodstream infection (BSI) isolates obtained over 9 years of observation. RESULTS: Whole-genome sequencing of 22 representative isolates showed (1) the presence of the φ3-prophage and diverse genetic features typical of animal-associated isolates (i.e., SCCmec XI element, Tn916 transposon and non φ3-prophages) in a majority of BSI isolates, (2) one BSI isolate devoid of the φ3-prophage but otherwise similar to an animal-infecting isolate, (3) 35 prophages carrying numerous genes previously associated with virulence or immune evasion in animal models of staphylococcal infections. The analysis of prophage content in all 76 BSI isolates showed an increasing prevalence of polylysogeny over time. Overall, over the course of the last 10 years, the BSI isolates appear to have acquired increasing numbers of genetic features previously shown to contribute to bacterial adaptation and virulence in animal models of staphylococcal infections. CONCLUSIONS: We hypothesize that lysogeny has played a significant role in increasing the ability of the ST398 clone to cause infections in humans. Our findings highlight the risk that the ST398 lineage will increase its threat to public health by continuing to acquire virulence and/or multiple antibiotic-resistance genes from hospital-associated clones of Staphylococcus aureus.

Emergence of blaNDM-7-Producing Enterobacteriaceae in Gabon, 2016.

Reports of carbapenemase-producing Enterobacteriaceae in Africa remain rare and assess mostly blaOXA-48-producing isolates from Mediterranean countries and South Africa. We identified blaNDM-7-producing Enterobacteriaceae in Gabon in 2016. The isolates contained blaNDM-7 IncX3 plasmids that were unusual and similar to the one described in a colistin-resistant Klebsiella pneumoniae SZ04 isolate from China.

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

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.

Comparative Genomics of Community-Associated Methicillin-Resistant Staphylococcus aureus Shows the Emergence of Clone ST8-USA300 in Geneva, Switzerland.

BACKGROUND: Previous investigations of community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) isolates have revealed a wide diversity of genetic backgrounds, with only sporadic occurrence of ST8-USA300, in Geneva, Switzerland. We conducted a molecular epidemiologic analysis to identify the origin of a sudden increase of ST8 PVL-positive isolates in Geneva during 2013. METHODS: On the basis of prospective CA-MRSA surveillance, we collected colonizing and infecting ST8-USA300 isolates and compared them to non-ST8 CA-MRSA isolates. Whole-genome sequencing (WGS) was performed for each isolate of this collection, and discriminating molecular features were linked to patient data. RESULTS: In 2013, 22 isolates with the ST8-USA300 profile were identified among 46 cases of CA-MRSA. WGS revealed 2 groups of strains that differed by the type of the SCCmec IV element encoded and whether they harbored an arginine catabolism mobile element (ACME) locus. ACME-negative strains were mainly isolated from patients traveling in or originating from South America. Single-nucleotide polymorphism positions in isolate groups were used to infer their common ancestor, determine their geographical origin, and trace their relatedness. CONCLUSIONS: WGS allowed the identification of transmission events and revealed that the increased prevalence of USA300 CA-MRSA isolates resulted from multiple importation events from the Americas but not from local clonal expansion of a successful clone.

Whole-genome sequence of Chryseobacterium oranimense, a colistin-resistant bacterium isolated from a cystic fibrosis patient in France.

For the first time, we report the whole-genome sequence analysis of Chryseobacterium oranimense G311, a multidrug-resistant bacterium, from a cystic fibrosis patient in France, including resistance to colistin. Whole-genome sequencing of C. oranimense G311 was performed using Ion Torrent PGM, and RAST, the EMBL-EBI server, and the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) database were used for annotation of all genes, including antibiotic resistance (AR) genes. General features of the C. oranimense G311 draft genome were compared to the other available genomes of Chryseobacterium gleum and Chryseobacterium sp. strain CF314. C. oranimense G311 was found to be resistant to all ß-lactams, including imipenem, and to colistin. The genome size of C. oranimense G311 is 4,457,049 bp in length, with 37.70% GC content. We found 27 AR genes in the genome, including ß-lactamase genes which showed little similarity to the known ß-lactamase genes and could likely be novel. We found the type I polyketide synthase operon followed by a zeaxanthin glycosyltransferase gene in the genome, which could impart the yellow pigmentation of the isolate. We located the O-antigen biosynthesis cluster, and we also discovered a novel capsular polysaccharide biosynthesis cluster. We also found known mutations in the orthologs of the pmrA (E8D), pmrB (L208F and P360Q), and lpxA (G68D) genes. We speculate that the presence of the capsular cluster and mutations in these genes could explain the resistance of this bacterium to colistin. We demonstrate that whole-genome sequencing was successfully applied to decipher the resistome of a multidrug resistance bacterium associated with cystic fibrosis patients.

The rhizome of the multidrug-resistant Enterobacter aerogenes genome reveals how new "killer bugs" are created because of a sympatric lifestyle.

Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of foreign languages.

Emergence of VIM-2 and IMP-15 carbapenemases and inactivation of oprD gene in carbapenem-resistant Pseudomonas aeruginosa clinical isolates from Lebanon.

We report here the emergence of VIM-2 and IMP-15 carbapenemases in a series of clinical isolates of carbapenem-resistant Pseudomonas aeruginosa in Lebanon. We also describe the disruption of the oprD gene by either mutations or insertion sequence (IS) elements ISPa1328 and ISPre2 isoform. Our study reemphasizes a rapid dissemination of the VIM-2 carbapenemase-encoding gene in clinical isolates of P. aeruginosa in the Mediterranean basin.

ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes.

ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) is a new bioinformatic tool that was created to detect existing and putative new antibiotic resistance (AR) genes in bacterial genomes. ARG-ANNOT uses a local BLAST program in Bio-Edit software that allows the user to analyze sequences without a Web interface. All AR genetic determinants were collected from published works and online resources; nucleotide and protein sequences were retrieved from the NCBI GenBank database. After building a database that includes 1,689 antibiotic resistance genes, the software was tested in a blind manner using 100 random sequences selected from the database to verify that the sensitivity and specificity were at 100% even when partial sequences were queried. Notably, BLAST analysis results obtained using the rmtF gene sequence (a new aminoglycoside-modifying enzyme gene sequence that is not included in the database) as a query revealed that the tool was able to link this sequence to short sequences (17 to 40 bp) found in other genes of the rmt family with significant E values. Finally, the analysis of 178 Acinetobacter baumannii and 20 Staphylococcus aureus genomes allowed the detection of a significantly higher number of AR genes than the Resfinder gene analyzer and 11 point mutations in target genes known to be associated with AR. The average time for the analysis of a genome was 3.35 ± 0.13 min. We have created a concise database for BLAST using a Bio-Edit interface that can detect AR genetic determinants in bacterial genomes and can rapidly and easily discover putative new AR genetic determinants.