Chemical Composition and Synergistic Potential of Mentha pulegium L. and Artemisia herba alba Asso. Essential Oils and Antibiotic against Multi-Drug Resistant Bacteria.

The essential oils were obtained by hydrodistillation from aerial parts of Mentha pulegium L. (M. pulegium L.) and Artemisia herba alba (A. herba alba) Asso. and analyzed by gas chromatography-flame ionization detector chromatograpy (GC-FID) and gaz chromatography-mass spectrometry (GC-MS). The antibacterial activities of the oils were determined by the disk diffusion method and a microdilution broth assay against six bacteria stains. The combinations of these essential oils with antibiotics were evaluated against two multi-drug-resistant bacteria strains: imipenem-resistant Acinetobacter baumannii (IRAB S3310) and methicillin-resistant Staphylococcus aureus (MRSA S19). The chemical analysis of M. pulegium essential oil revealed the presence of pulegone (74.8%) and neoisomenthol (10.0%). A. herba alba essential oil was characterized by camphor (32.0%), α-thujone (13.7%), 1,8-cineole (9.8%), ß-thujone (5.0%), bornéol (3.8%), camphene (3.6%), and p-cymene (2.1%). All strains tested except Pseudomonas aeruginosa were susceptible to these oils. The combinations of essential oils with antibiotics exerted synergism, antagonism, or indifferent effects. The best effect was observed with A. herba alba essential oil in association with cefoxitin (CX) against MRSA S19. However, for IRAB S3310, the strongest synergistic effect was observed with M. pulegium in association with amikacin (AK). This study demonstrated that M. pulegium and A. herba alba essential oils have antibacterial activities which could be potentiated by antibiotics especially in the case of IRAB S3310.

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.

Co-occurrence of carbapenemase encoding genes in Acinetobacter baumannii, a dream or reality?

BACKGROUND: Acinetobacter baumannii is an important opportunistic pathogen that is rapidly evolving towards multidrug resistance and is responsible for life-threatening infections. Carbapenems are commonly used to treat A. baumannii infections but the emergence of carbapenemase encoding genes, such as blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM has been reported. Moreover, several studies have reported the co-occurrence of two distinct carbapenemases in some isolates. The aim of the present study is to demonstrate whether the phenomenon of co-occurrence of two distinct carbapenemase encoding genes in a single isolate still exists. RESULTS: We studied six strains of A. baumannii including one harboring blaOXA-23-like and blaOXA-24-like genes and five with blaOXA-23-like and blaNDM genes. One colony of each strain was inoculated in sterile water and diluted ten-fold. Each dilution was cultivated on trypticase soy agar plates for 24 h at 37 °C and the isolated bacteria were analyzed. For two of the six tested strains, we identified two different populations of A. baumannii, each with a different carbapenemase, genes encoding aminoglycoside modifying enzymes, resistance phenotype, and clonal type. In addition, the two different populations had the same aspect on the agar plate. CONCLUSIONS: Here, we demonstrate that A. baumannii infections could be linked to multiple clones harboring different carbapenemase encoding genes in the same sample. In addition, we describe an easy method of verifying the presence of co-occurrence of carbapenemase in one isolate.

Molecular Tests That Target the RTX Locus Do Not Distinguish between Kingella kingae and the Recently Described Kingella negevensis Species.

Kingella kingae is an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis of K. kingae infections. However, the present study shows that Kingella negevensis, a Kingella species newly identified in young children, harbors an identical Kingella RTX locus, raising the question of whether K. negevensis can be misidentified as K. kingae by clinical microbiology laboratories. In silico comparison of Kingella sp. RTX and groEL genes and in vitro studies provided evidence that targeting the rtxA and rtxB genes could not differentiate between strains of K. kingae and K. negevensis, whereas targeting the groEL gene could. This prompted the design of a highly specific and sensitive qPCR assay targeting K. negevensis groEL (kngroEL). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay and Kingella-specific rtxB, K. kingae-specific groEL (kkgroEL), and kngroEL qPCR assays. Forty-two specimens were rtxB positive, including 41 that were also kkgroEL positive and 1 (the remaining one) that was kngroEL positive. Thus, this study discloses an invasive infection caused by K. negevensis in humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis of K. kingae infections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused by K. negevensis in humans.

Prevalence and emergence of carbapenemases-producing Gram-negative bacteria in Mediterranean basin.

The emergence and the global spread of carbapenemases concern to health services worldwide. Their celestial rise among Gram-negative bacilli has challenged both the scientific and pharmaceutical sectors. Indeed, infections caused by these bacteria have limited treatment options and have been associated with high mortality and morbidity rates. Carbapenemase producers are mainly identified among Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii and still mostly in hospital settings and rarely in the community. They are closely related to KPC, VIM, IMP, NDM, and OXA-48 types. The encoding genes are mostly plasmid located and associated with various mobile genetic elements. The Mediterranean area is of interest due to a great diversity and population mixing. The prevalence of carbapenemases is particularly high and variant among countries, partially depending on the population exchange relationship between the regions and the possible reservoirs of each carbapenemase. This review described the epidemiology of carbapenemases in this region of the world highlighting the worrisome situation and the need to screen and detect these enzymes to prevent and control their dissemination especially as it is clear that very few novel antibiotics will be introduced in the next few years, making the dissemination of carbapenem-resistant Gram-negative bacteria of crucial importance worldwide.

Isolation and characterization of Kingella negevensis sp. nov., a novel Kingella species detected in a healthy paediatric population.

We herein report the isolation and characterization of 21 Gram-stain-negative strains cultivated from the oropharynx of healthy children in Israel and Switzerland. Initially described as small colony variants of Kingella kingae, phenotypic analysis, biochemical analysis, phylogenetic analysis based on sequencing of the partial 16S rRNA gene and five housekeeping genes (abcZ, adk, G6PD, groEL and recA), and whole genome sequencing and comparison between members of the genera Kingella and Neisseria provided evidence for assigning them to the genus Kingella. Cellular fatty acids included important amounts of C12 : 0, C14 : 0, C16 : 0 and C16 : 1n7. Digital DNA-DNA hybridization between the isolates Sch538T and K. kingae ATCC 23330T revealed relatedness of 19.9 %. Comparative analysis of 16S rRNA gene sequences available in GenBank allowed matches to strains isolated in the USA, suggesting a wider geographical distribution. A novel species named Kingella negevensis sp. nov. is proposed, as most strains have been isolated in the Negev, a desert region of southern Israel. The type strain is Sch538T (=CCUG 69806T=CSUR P957).

Prevalence and clonal relationship of ESBL-producing Salmonella strains from humans and poultry in northeastern Algeria.

BACKGROUND: The aims of this study were to investigate Salmonella contamination in broiler chicken farms and slaughterhouses, to assess the antibiotic resistance profile in avian and human Salmonella isolates, and to evaluate the relationship between avian and human Extended Spectrum ß-Lactamase (ESBL)-producing isolates. Salmonella was screened in different sample matrices collected at thirty-two chicken farms and five slaughterhouses. The human isolates were recovered from clinical specimens at the University Teaching Hospital of Constantine (UTH). All suspected colonies were confirmed by MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time OF light) and serotyped. Susceptibility testing against 13 antibiotics including, amoxicillin/clavulanic acid, ticarcillin, cefoxitin, cefotaxime, aztreonam, imipenem, ertapenem, gentamicin, amikacin, ciprofloxacin, colistin, trimethoprim/sulfamethoxazole and fosfomycin, was performed using the disk diffusion method on Mueller-Hinton agar. ESBL-production was screened by the double-disk synergy test and confirmed by molecular characterization using PCR (polymerase chain reaction) amplification and sequencing of ESBL encoding genes. Clonality of the avian and human strains was performed using the Multi Locus Sequencing Typing method (MLST). RESULTS: Forty-five isolated avian Salmonella strains and 37 human collected ones were studied. Five S. enterica serotypes were found in avian isolates (mainly Kentucky) and 9 from human ones (essentially Infantis). 51.11% and 26.6% of the avian isolates were resistant to ciprofloxacin and cefotaxime, respectively, whereas human isolates were less resistant to these antibiotics (13.5% to ciprofloxacin and 16.2% to cefotaxime). Eighteen (12 avian and 6 human) strains were found to produce ESBLs, which were identified as bla CTX-M-1 (n = 12), bla CTX-M-15 (n = 5) and bla TEM group (n = 8). Interestingly, seven of the ESBL-producing strains (5 avian and 2 human) were of the same ST (ST15) and clustered together, suggesting a common origin. CONCLUSION: The results of the combined phenotypic and genotypic analysis found in this study suggest a close relationship between human and avian strains and support the hypothesis that poultry production may play a role in the spread of multidrug-resistant Salmonella in the human community within the study region.

The impact of culturomics on taxonomy in clinical microbiology.

Over the past decade, new culture methods coupled to genome and metagenome sequencing have enabled the number of isolated bacterial species with standing in nomenclature to rise to more than 15,000 whereas it was only 1791 in 1980. 'Culturomics', a new approach based on the diversification of culture conditions, has enabled the isolation of more than 1000 distinct human-associated bacterial species since 2012, including 247 new species. This strategy was demonstrated to be complementary to metagenome sequencing for the exhaustive study of the human microbiota and its roles in health and diseases. However, by identifying a large number of new bacterial species in a short time, culturomics has highlighted a need for taxonomic approaches adapted to clinical microbiology that would include the use of modern and reproducible tools, including high throughput genomic and proteomic analyses. Herein, we review the development of culturomics and genomics in the clinical microbiology field and their impact on bacterial taxonomy.

Description of strain FC3T as the neotype strain of Actinobaculum massiliense.

Actinobaculum massiliense (Euzéby, 2006) was isolated from the urine of an elderly woman in 2001. Unfortunately, the strain deposited as the type strain was, by error, an Actinobaculum schaalii strain (Yassin et al., 2015). In 2015, we isolated a new strain of A. massiliense, FC3, from the urine of a 12-year-old patient with acute cystitis. We herein present the characteristics of strain FC3 (=CSUR P1982=DSM 100580) and formally propose it as the neotype strain of A. massiliense.

High prevalence of bla(NDM-1) carbapenemase-encoding gene and 16S rRNA armA methyltransferase gene among Acinetobacter baumannii clinical Isolates in Egypt.

The main objective of this study was to decipher the molecular mechanism of resistance to carbapenems and aminoglycosides in a large series of 150 Acinetobacter baumannii clinical isolates collected from July 2012 to September 2013 in Egypt. We report for the first time the emergence of bla(NDM-1) and the cooccurrence of 16S rRNA methylase armA with bla(NDM-1) and bla(OXA-23) in Egyptian hospitals. Multilocus sequence typing identified 27 distinct sequence types, 11 of which were novel.

First report of 16S rRNA methylase ArmA-producing Acinetobacter baumannii and rapid spread of metallo-ß-lactamase NDM-1 in Algerian hospitals.

PURPOSE: The aim of the present study was to characterize the molecular support of resistance to carbapenems, aminoglycosides and fluoroquinolones in carbapenem-resistant Acinetobacter baumannii clinical isolates recovered between January 2011 and April 2013 from Algerian hospitals. METHODS: Antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Carbapenemase activity was detected using both MALDI-TOF mass spectrometry assay and via microbiological tests. Carbapenem, aminoglycoside and fluoroquinolone resistance determinants were studied by PCR and sequencing. Clonal relationships between strains were determined using Multi Locus Sequence Typing (MLST). RESULTS: A total of 47 imipenem-resistant A. baumannii were isolated and identified by MALDI-TOF mass spectrometry. All imipenem-resistant strains were positive in the modified Hodge test, and EDTA inhibited the activity of metallo-ß-lactamases enzymes in 11 strains. The blaOXA-23 gene was detected in 33 strains and the blaOXA-24 gene in 10 strains. The metallo-ß-lactamase blaNDM-1 gene was detected in 11 isolates (23.4%) from Algiers and Sétif, including 7 that co-expressed a blaOXA-23 gene. Resistance to aminoglycosides was due to the production of aminoglycoside-modifying enzymes, AAC(3)-Ia, AADA, ANT(2″)-I, APH(3')-VI, and 16S rRNA methylases, ArmA. The fluoroquinolone resistance was mainly associated with mutations at Ser83Leu and Ser80Leu of the gyrA and parC genes, respectively. MLST revealed five sequence types (STs), 1, 2, 19, 25, and 85. The imipenem-resistant A. baumannii ST2 was the predominant clone (35/47). CONCLUSIONS: Here we report for the first time clinical multidrug-resistant A. baumannii isolates harboring 16S rRNA methylase gene, armA, and rapid spread of metallo-ß-lactamase NDM-1 isolated from patients in Algeria.

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.

Spread of OXA-48 and NDM-1-Producing Klebsiella pneumoniae ST48 and ST101 in Chicken Meat in Western Algeria.

Aim: We investigated the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in chicken meat in Western Algeria in 2017. Results: From February to July 2017, samples of chicken meat from three poultry farms in Western Algeria were screened for the presence of CPE. Strains were characterized with regard to antibiotic resistance, ß-lactamase content, Plasmid-mediated quinolone resistance, sulfonamide resistance genes, clonality (repetitive sequence-based profiles and multilocus sequence typing) and virulence traits. Of 181 samples analyzed, 29 (16.0%) carbapenemase-producing Klebsiella pneumoniae were detected. Twenty-three OXA-48-producers (79.3%) and six (20.7%) New Delhi metallo (NDM)-1-producers were observed. Clonality analysis showed three distinct lineages and clonal expansions of the OXA-48-producing K. pneumoniae ST48 and the NDM-1-producing K. pneumoniae ST101. These isolates harbored fimH, ureA, mrkD, entB, uge, and wabG. Neither capsular serotype genes nor hypermucoviscous phenotype were detected. Plasmid analysis confirmed that all these isolates harbored the transferable IncL and IncFIIK plasmids. Conclusions: This study reports the spread of OXA-48 and NDM-1-producing K. pneumoniae ST48 and ST101 in chicken meat in Western Algeria and demonstrates that food represents a reservoir of the carbapenemases encoding genes.

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.

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.

Characterisation of blaOXA-538, a new variant of blaOXA-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.

Incidence of OXA-23 and OXA-58 Carbapenemases Coexpressed in Clinical Isolates of Acinetobacter baumannii in Tunisia.

Acinetobacter baumannii is an important opportunistic and multidrug-resistant pathogen responsible for nosocomial infections in health facilities. The aim of this study was to characterize the molecular mechanisms of carbapenem resistance in A. baumannii isolates isolated from Mohamed Kassab Orthopedic Institute in Tunis, Tunisia. Twenty-five imipenem-resistant A. baumannii clinical isolates collected between 2013 and 2016 were identified using API 20NE and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase activity was detected using microbiological tests and PCR. The epidemiological relatedness of the isolates was studied using multilocus sequence typing (MLST). The isolates were resistant to all antibiotics tested with increased minimum inhibitory concentration values (>32 mg/L). The microbiological tests showed that the 25 A. baumannii were positive for modified Hodge test and for the Carba NP test; however, ß-lactamase activity was not inhibited by EDTA. All the isolates harbored the naturally occurring blaOXA-51-like gene and the blaOXA-23-like carbapenemase gene. Among these isolates, one isolate coexpressed the blaOXA-58 gene. MLST revealed several sequence types (STs) with the predominance of ST2 imipenem-resistant A. baumannii (14/25; 56%). In this study we report the prevalence of ST2 imipenem resistance and for the first time the coexpression of blaOXA-23 and blaOXA-58 in clinical isolates of A. baumannii in a Tunisian hospital.