Dissemination of Carbapenemases and MCR-1 Producing Gram-Negative Bacteria in Aquatic Environments in Batna, Algeria.

Antibiotic-resistant-bacteria are being considered as emerging environmental contaminants where the importance of the surrounding environment in their emergence and dissemination has been emphasized. The aim of this study was to screen for the presence and diversity of carbapenem- and colistin-resistant Gram-negative bacteria (GNBs) in different aquatic environments. Water samples were collected in Batna, Algeria. Carbapenem- and colistin-resistant GNBs were selectively isolated and then identified using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. After phenotypic antibiotic susceptibility testing, the molecular mechanisms of ß-lactams and colistin-resistance were investigated by PCR and sequencing. The clonality of mcr-1 positive Escherichia coli was determined by multi-locus sequence typing. We noticed a high level of resistance in both tap water and wastewater. The most commonly found carbapenem-resistance mechanism was the OXA-48 enzyme, but other carbapenemases were also detected. In addition, the mcr-1 gene was detected in 18 E. coli of different sequence types. Our findings highlight the role of aquatic environments in the dissemination of resistant-bacteria, especially considering that water is a connecting medium between different ecological systems and can easily transmit resistant-bacteria and promote horizontal gene transfer. Thus, the development of effective treatment strategies for eliminating antibiotic-resistance is seriously needed.

Miscarriage Risk Factors for Pregnant Women: A Cohort Study in Eastern Algeria's Population.

Background: Miscarriage is defined as an adverse and unexpected termination of pregnancy; it is the most frequent pregnancy complication. Here, we aimed to identify the factors predisposing to miscarriage in pregnant women in Eastern Algeria and the effect of the combination of several factors, including maternal Body Mass Index (BMI), maternal age, concomitant pathologies, and nutrients, and to predict the occurrence of miscarriage. Methods: A total of 786 pregnant women from Eastern Algeria were interviewed between 2011 and 2015. Association between miscarriage exposure and identified risk factors was assessed using a Generalized Linear Model (GLM), ANOVA test, Multiple Correspondence Analysis (MCA), and Hierarchical Clustering Analysis (HCA). Throughout this study, we sought to find answers, discuss this association, and predict the occurrence of miscarriage. Results: We developed a predictive model for miscarriage, and we found that miscarriage was significantly higher for pregnant women aged over 35 years (1.75; 95% CI: 0.75-4.37; p = 0.208), with a high BMI (> 25 kg/m2), (1.88; 95% CI:1.28-2.78; p = 0.001). We have highlighted that miscarriage is strongly associated with hypertension (1.67; 95% CI: 1.16-2.39; p = 0.006), diet rich in meat (0.60; 95% CI: 0.33-1.04; p = 0.075), and moderate in fish (2.32; 95% CI: 1.18-4.58; p = 0.015). Conclusion: Our study proved that knowing these risk factors helps to establish predictive models and strategies to prevent tragic pregnancy outcomes and highlights the link between miscarriage and several risk factors; and thus, will allow protecting mother and fetus health.

Occurrence of Extended Spectrum Cephalosporin-, Carbapenem- and Colistin-Resistant Gram-Negative Bacteria in Fresh Vegetables, an Increasing Human Health Concern in Algeria.

The aim of this study was to screen for extended spectrum cephalosporin-, carbapenem- and colistin-resistant Gram-negative bacteria in fresh vegetables in Batna, Algeria. A total of 400 samples of fresh vegetables were collected from different retail stores. Samples were immediately subjected to selective isolation, then the representative colonies were identified using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Phenotypic and genotypic analyses were carried out in terms of species identification and relative antibiotic resistance. Transferability of the carbapenemase and mcr-bearing plasmids was verified by conjugation. The clonal relationships of carbapenemase and mcr-positive Escherichia coli isolates were studied by multi-locus sequence typing (MLST). Sixty-seven isolates were characterised and were mostly isolated from green leafy vegetables, where the dominant species identified included Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomona maltophilia, E. coli and Citrobacter braakii. PCR and sequencing results showed that E. coli was the bacterial species presenting the highest antibiotic resistance level in parallel to blaTEM (n = 16) and blaCTX-M-15 (n = 11), which were the most detected genes. Moreover, five isolates carried carbapenemase genes, including the blaOXA-48 and/or blaVIM-4 genes. The mcr-1 gene was detected in two E. coli isolates. MLST analysis revealed three different E. coli sequence types: ST101 (n = 1), ST216 (n = 1) and ST2298 (n = 1). Conjugation assays confirmed the transferability of the blaOXA-48 and mcr-1 genes. In this study we report, for the first time, the detection of the blaOXA-48 gene in E. coli and C. braakii isolates and the blaVIM-4 gene in vegetables. To the best of our knowledge, this is the first report on the detection of mcr-1 genes from vegetables in Algeria.

Dissemination of OXA-48- and NDM-1-Producing Enterobacterales Isolates in an Algerian Hospital.

Multidrug-resistant (MDR) Enterobacterales remain an increasing problem in Algeria, notably due to the emergence of carbapenemase producers. We investigated the molecular characteristics of carbapenem-resistant Enterobacterales isolates recovered from outpatients and inpatients in Eastern Algeria. Non-repetitive Enterobacterales with reduced susceptibility to carbapenems were consecutively collected from clinical specimens in Annaba University Hospital (Algeria) between April 2016 and December 2018. Isolates were characterized with regard to antibiotic resistance, resistome and virulome content, clonality, and plasmid support. Of the 168 isolates analyzed, 29 (17.3%) were carbapenemase producers and identified as K. pneumoniae (n = 23), E. coli (n = 5), and E. cloacae (n = 1). blaOXA-48 was the most prevalent carbapenemase-encoding gene (n = 26/29), followed by blaNDM-1 gene (n = 3/29). K. pneumoniae isolates harbored some virulence traits (entB, ugeF, ureA, mrkD, fimH), whereas E. coli had a commensal origin (E, A, and B1). Clonality analysis revealed clonal expansions of ST101 K. pneumoniae and ST758 E. coli. Plasmid analysis showed a large diversity of incompatibility groups, with a predominance of IncM (n = 26, 89.7%). A global dissemination of OXA-48-producing Enterobacterales in the Algerian hospital but also the detection of NDM-1-producing E. coli in community settings were observed. The importance of this diffusion must be absolutely investigated and controlled.

Detection of blaOXA-48 and mcr-1 Genes in Escherichia coli Isolates from Pigeon (Columba livia) in Algeria.

The emergence and spread of ß-lactams and colistin-resistant Escherichia coli in birds deserve a special concern worldwide. This study aimed to investigate the presence of ß-lactams and colistin-resistant Escherichia coli strains isolated from the faeces of urban and rural pigeons in Batna, Algeria, and to characterise their molecular traits of resistance. Between March and April 2019, a total of 276 faecal droppings samples were collected in Batna, Algeria. Samples were subjected to selective isolation of ß-lactams and colistin-resistant Escherichia coli. The representative colonies were then identified using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. Antimicrobial susceptibility testing was performed using the disc diffusion method. ß-lactamases, as well as mcr genes, were screened for by PCR and confirmed by sequencing. Genetic relatedness of the mcr-positive E. coli strains was determined using multi-locus sequence typing analysis. Transferability features of carbapenemase genes were assessed by conjugation experiments. Overall, thirty-five E. coli isolates were obtained only from urban pigeon samples. All carbapenem-resistant isolates harboured the blaOXA-48 gene as the only carbapenemase gene detected (n = 11), while blaESBL genes were detected in eighteen isolates. Out of the thirty-five isolates, four E. coli isolates were positive for the mcr-1 gene. The obtained mcr-1 positive E. coli isolates belonged to four STs, including ST1485, ST224, ST46, and a new ST. This study is the first to report the isolation of E. coli strains carrying the mcr-1 gene from pigeon faeces in Algeria and also the first to report the detection of blaOXA-48-positive E. coli in pigeons. Close surveillance is, therefore, urgently needed to monitor the dissemination of blaOXA-48 and mcr-1 producing E. coli strains in wildlife.

Detection of NDM-5 and MCR-1 antibiotic resistance encoding genes in Enterobacterales in long-distance migratory bird species Ciconia ciconia, Algeria.

ß-lactams and colistin resistance in Enterobacterales is a global public health issue. In this study we aimed to investigate the occurrence and genetic determinants of Extended-Spectrum ß-lactamases, carbapenemases and mcr-encoding-genes in Enterobacterales isolates recovered from the migratory bird species Ciconia ciconia in an Algerian city. A total of 62 faecal samples from white storks were collected. Samples were then subjected to selective isolation of ß-lactams and colistin-resistant-Enterobacterales. The representative colonies were identified using Matrix-Assisted Laser Desorption-Ionisation Time-of-Flight Mass Spectrometry. Susceptibility testing was performed using the disk-diffusion method. ESBL, carbapenemases, and colistin resistance determinants were searched for by PCR and sequencing. The clonality relationships of the obtained isolates were investigated by multilocus sequence typing assays. Mating experiments were carried out to evaluate the transferability of the carbapenemase and mcr-genes. Forty-two isolates were identified as follows: Escherichia coli (n = 33), Klebsiella pneumoniae (n = 4), Proteus mirabilis (n = 4) and Citrobacter freundii (n = 1). Molecular analysis showed that twelve isolates carried the blaESBL genes alone, fifteen E. coli isolates were positive for the blaOXA-48 gene, six isolates were NDM-5-carriers (two P. mirabilis, two K. pneumoniae and two E. coli) and eight E. coli strains were positive for the mcr-1 gene. MLST results showed a high clonal diversity, where NDM-5-producing strains were assigned to two sequence types (ST167 for E. coli and ST198 for K. pneumoniae), whereas the mcr-1 positive E. coli isolates belonged to ST58, ST224, ST453, ST1286, ST2973, ST5542, ST9815 and the international high-risk resistant lineage ST101. To the best of our knowledge, this is the first report of blaNDM-5 gene in white storks and also the first describing the mcr-1 gene in white storks in Algeria. This study underlines the important role of migratory white storks as carriers of high-level drug-resistant bacteria, allowing their possible implication as indicators and sentinels for antimicrobial resistance surveillance.

Emergence of Metallo-ß-Lactamases and OXA-48 Carbapenemase Producing Gram-Negative Bacteria in Hospital Wastewater in Algeria: A Potential Dissemination Pathway Into the Environment.

Antibiotic-resistant bacteria can leave hospitals and therefore contaminate the environment and, most likely, humans and animals, through different routes, among which wastewater discharge is of great importance. This study aims to assess the possible role of hospital sewage as reservoir and dissemination pathway of carbapenem-resistant Gram-negative bacilli (GNB). Carbapenem-resistant GNB were selectively isolated from wastewater collected from a public hospital in Batna, Algeria. Species identification was carried out using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry, and antibiotic susceptibility was evaluated by the disc diffusion method. ß-Lactamase production was investigated phenotypically using the double-disk synergy assay and the modified CarbaNP test, then the molecular mechanisms of ß-lactam-resistance were studied by PCR and sequencing. Ten Enterobacteriaceae and 14 glucose-nonfermenting GNB isolates were obtained. All Enterobacteriaceae isolates were positive for OXA-48 and TEM-1D ß-lactamases, where seven of them coproduced an extended-spectrum ß-lactamase. VIM-2 carbapenemase was detected in six glucose-nonfermenting GNB isolates. However, three Pseudomonas aeruginosa, one Comamonas jiangduensis and one Acinetobacter baumannii isolates were positive for VIM-4 variant. In addition, NDM-1 enzyme was detected in four A. baumannii isolates. Our findings highlight the potential impact of hospital wastewater in the spread of drug resistance mechanisms outside of hospitals.

Vegetables and Fruit as a Reservoir of ß-Lactam and Colistin-Resistant Gram-Negative Bacteria: A Review.

Antibacterial resistance is one of the 2019 World Health Organization's top ten threats to public health worldwide. Hence, the emergence of ß-lactam and colistin resistance among Gram-negative bacteria has become a serious concern. The reservoirs for such bacteria are increasing not only in hospital settings but in several other sources, including vegetables and fruit. In recent years, fresh produce gained important attention due to its consumption in healthy diets combined with a low energy density. However, since fresh produce is often consumed raw, it may also be a source of foodborne disease and a reservoir for antibiotic resistant Gram-negative bacteria including those producing extended-spectrum ß-lactamase, cephalosporinase and carbapenemase enzymes, as well as those harboring the plasmid-mediated colistin resistance (mcr) gene. This review aims to provide an overview of the currently available scientific literature on the presence of extended-spectrum ß-lactamases, cephalosporinase, carbapenemase and mcr genes in Gram-negative bacteria in vegetables and fruit with a focus on the possible contamination pathways in fresh produce.

MCR-5-Producing Colistin-Resistant Cupriavidus gilardii Strain from Well Water in Batna, Algeria.

This paper presents the first description of the mcr-5.1 gene in a colistin-resistant Cupriavidus gilardii isolate from well water that supplies a maternity hospital in Algeria. The whole-genome sequence of this strain showed the presence of putative ß-lactamase, aac(3)-IVa, and multidrug efflux pump-encoding genes, which could explain the observed multidrug resistance phenotype. Our findings are of great interest, as we highlight a potential contamination route for the spread of mcr genes. IMPORTANCE Colistin resistance mediated by mcr genes in Gram-negative bacteria has gained significant attention worldwide. This is due to the ability of these genes to be horizontally transferred between different bacterial genera and species. Aquatic environments have been suggested to play an important role in the emergence and spread of this resistance mechanism. Here, we describe the first report of an mcr-5-positive Cupriavidus gilardii aquatic isolate through its isolation from well water in Algeria. The significance of our study is in shedding the light on an important environmental reservoir of mcr genes.

Epidemiology of mobile colistin resistance (mcr) genes in aquatic environments.

Colistin is one of the last-line therapies against multidrug-resistant Gram-negative pathogens, especially carbapenemase-producing isolates, making resistance to this compound a major global public-health crisis. Until recently, colistin resistance in Gram-negative bacteria was known to arise only by chromosomal mutations. However, a plasmid-mediated colistin resistance mechanism was described in late 2015. This mechanism is encoded by different mobile colistin resistance (mcr) genes that encode phosphoethanolamine (pEtN) transferases. These enzymes catalyse the addition of a pEtN moiety to lipid A in the bacterial outer membrane leading to colistin resistance. MCR-producing Gram-negative bacteria have been largely disseminated worldwide. However, their environmental dissemination has been underestimated. Indeed, water environments act as a connecting medium between different environments, allowing them to play a crucial role in the spread of antibiotic resistance between the natural environment and humans and other animals. For a better understanding of the role of such environments as reservoirs and/or dissemination routes of mcr genes, this review discusses primarily the various water habitats contributing to the spread of antibiotic resistance. Thereafter, we provide an overview of existing knowledge regarding the global epidemiology of mcr genes in water environments. This review confirms the global distribution of mcr genes in several water environments, including wastewater from different origins, surface water and tap water, making these environments reservoirs and dissemination routes of concern for this resistance mechanism.

Carbapenemase-producing Gram-negative bacteria in aquatic environments: a review.

Antibiotic resistance is one of the greatest public-health challenges worldwide, especially with regard to Gram-negative bacteria (GNB). Carbapenems are the ß-lactam antibiotics of choice with the broadest spectrum of activity and, in many cases, are the last-resort treatment for several bacterial infections. Carbapenemase-encoding genes, mainly carried by mobile genetic elements, are the main mechanism of resistance against carbapenems in GNB. These enzymes exhibit a versatile hydrolytic capacity and confer resistance to most ß-lactam antibiotics. After being considered a clinical issue, increasing attention is being giving to the dissemination of such resistance mechanisms in the environment and especially through water. Aquatic environments are among the most significant microbial habitats on our planet, known as a favourable medium for antibiotic gene transfer, and they play a crucial role in the huge spread of drug resistance in the environment and the community. In this review, we present current knowledge regarding the spread of carbapenemase-producing isolates in different aquatic environments, which may help the implementation of control and prevention strategies against the spread of such dangerous resistant agents in the environment.

First detection of an OXA-48-producing Enterobacter cloacae isolate from currency coins in Algeria.

OBJECTIVES: The aim of this study was to screen for the presence of ß-lactamase-producing Gram-negative bacteria (GNB) from Algerian currency collected from food vendors in Batna city, Algeria. METHODS: During two periods (May 2018 and March-April 2019), a total of 408 coins and currency notes of different denominations of Algerian Dinar were randomly recovered from several food vendors. Samples were subjected to selective isolation of extended-spectrum cephalosporin- and carbapenem-resistant GNB. Bacterial species identification was performed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was performed by the disk diffusion method. Carbapenemase and extended-spectrum ß-lactamase (ESBL) genes were searched for by real-time PCR, standard PCR and sequencing. The clonal relationship of carbapenemase-producing isolates was investigated by multilocus sequence typing (MLST). The transferability of the detected carbapenemase-encoding gene was verified by conjugation experiments. RESULTS: Twelve cefotaxime- and/or carbapenem-resistant strains were isolated in this study and were identified as Enterobacter cloacae, Raoultella ornithinolytica, Citrobacter freundii, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas libanensis and Pseudomonas stutzeri. The blaOXA-48 gene was detected in only one E. cloacae strain belonging to sequence type 108 (ST108), whilst the two R. ornithinolytica isolates harboured blaCTX-M-27 and one E. coli strain carried blaCTX-M-14. The detected blaOXA-48 gene was transferable by conjugation. CONCLUSIONS: We report for the first time the detection of an OXA-48-producing E. cloacae isolate from money. This calls for consciousness development on the potential risks associated with poor handling of currency.

Petroleum degradation by endophytic Streptomyces spp. isolated from plants grown in contaminated soil of southern Algeria.

Petroleum hydrocarbons are well known by their high toxicity and recalcitrant properties. Their increasing utilization around worldwide led to environmental contamination. Phytoremediation using plant-associated microbe is an interesting approach for petroleum degradation and actinobacteria have a great potential for that. For this purpose, our study aimed to isolate, characterize, and assess the ability of endophytic actinobacteria to degrade crude petroleum, as well as to produce plant growth promoting traits. Seventeen endophytic actinobacteria were isolated from roots of plants grown naturally in sandy contaminated soil. Among them, six isolates were selected on the basis of their tolerance to petroleum on solid minimal medium and characterized by 16S rDNA gene sequencing. All petroleum-tolerant isolates belonged to the Streptomyces genus. Determination by crude oil degradation by gas chromatorgraph-flame ionization detector revealed that five strains could use petroleum as sole carbon and energy source and the petroleum removal achieved up to 98% after 7 days of incubation. These isolates displayed an important role in the degradation of the n-alkanes (C6-C30), aromatic and polycyclic aromatic hydrocarbons. All strains showed a wide range of plant growth promoting features such as siderophores, phosphate solubilization, 1-aminocyclopropane-1-carboxylate deaminase, nitrogen fixation and indole-3-acetic acid production as well as biosurfactant production. This is the first study highlighting the petroleum degradation ability and plant growth promoting attributes of endophytic Streptomyces. The finding suggests that the endophytic actinobacteria isolated are promising candidates for improving phytoremediation efficiency of petroleum contaminated soil.

Migratory White Stork (Ciconia ciconia): A Potential Vector of the OXA-48-Producing Escherichia coli ST38 Clone in Algeria.

The emergence of carbapenemase-producing Enterobacteriaceae is of great concern to public health worldwide. The aim of this study was to screen for the presence of carbapenemase-producing Enterobacteriaceae in white stork (Ciconia ciconia) migratory bird stools, and to investigate their molecular support on ß-lactamase production. In March 2015, 32 fecal samples of white stork were collected in the Commune of El Madher Wilaya de Batna, in eastern Algeria. Samples were subjected to selective isolation of carbapenem-resistant Enterobacteriaceae. Representative colonies were screened phenotypically for carbapenemase production. Carbapenemase-producing isolates were subjected to antibiotic susceptibility testing and extended-spectrum ß-lactamase (ESBL) coproduction. ß-Lactamase determinants were searched for by PCR and sequencing. Three carbapenemase-producing Escherichia coli were obtained. Only one strain was positive for ESBL production. The OXA-48-type carbapenemase-encoding gene was detected in all isolates. Screening for other ß-lactamase-encoding genes showed that all isolates coexpress the blaTEM gene, whereas one of them additionally harbored the blaCTX-M-15 ESBL gene. Multilocus sequence typing results showed that two strains belonged to the sequence type 38. This work demonstrated for the first time that the migratory white stork can play an important role in the dissemination of OXA-48-producing E. coli as a potential reservoir and vector.

Molecular characterisation of carbapenemases in urban pigeon droppings in France and Algeria.

OBJECTIVES: The main objective of this study was to detect the presence of carbapenemase-encoding genes in stool samples of urban pigeons. METHODS: Stool samples were collected from 73 pigeons in two Mediterranean cities, namely Marseille (France) and Annaba (Algeria). Faecal samples were screened by real-time PCR and standard PCR for the presence of carbapenemase-encoding genes. RESULTS: Carbapenem resistance genes were detected in 16 (21.9%) of the samples, with 8 positive for blaOXA-23, 12 positive for blaOXA-51-like and 13 positive for blaOXA-58. No samples were positive for blaNDM-1, blaOXA-24, blaOXA-48, blaVIM or blaKPC. All positive samples were screened for the presence of Acinetobacter spp. by partial rpoB gene sequence amplification, and the results showed the presence of five Acinetobacter spp., with percentage similarities to related species in GenBank ranging between 96% and 100%. The dominant species was Acinetobacter guillouiae, followed by Acinetobacter baumannii, Acinetobacter haemolyticus, Acinetobacter pittii and Acinetobacter nosocomialis. One DNA sequence showed a very low degree of homology (92%) with Acinetobacter gerneri, suggesting a new Acinetobacter spp. CONCLUSIONS: Here we report the first detection of carbapenemase-encoding genes from urban pigeon stools. These results question the potential of birds as a reservoir for the spread of these resistance determinants both in animals and humans.

Draft Genome Sequence of Streptomyces specialis Type Strain GW41-1564 (DSM 41924).

Here, we report the draft genome sequence of Streptomyces specialis type strain GW41-1564, which was isolated from soil. This 5.87-Mb genome exhibits a high G+C content of 72.72% and contains 5,486 protein-coding genes.

Outbreak of OXA-48-Producing Klebsiella pneumoniae Involving a Sequence Type 101 Clone in Batna University Hospital, Algeria.

Seven nonredundant ertapenem-resistant Klebsiella pneumoniae isolates were collected between May 2014 and 19 January 2015 in the nephrology and hematology units of Batna University Hospital in Algeria. All strains coproduced the blaOXA-48, blaCTX-M-15, blaSHV-1, and blaTEM-1D genes. Six of these isolates belonged to the pandemic clone sequence type 101 (ST101). The blaOXA-48 gene was located on a conjugative IncL/M-type plasmid. This is the first known outbreak of OXA-48-producing K. pneumoniae isolates involving an ST101 clone in Batna University Hospital.