Prevalence and Genomic Investigation of Salmonella Isolates Associated with Watermelons and Their Environmental Reservoirs in Bejaia, Algeria.

This study was conducted in Bejaia, Algeria, to determine the presence of Salmonella in fresh watermelon (n = 105), soil (n = 23), and irrigation water samples (n = 17) collected from two different farms. After isolation, antimicrobial susceptibility testing, serotype determination, multilocus sequence typing, antimicrobial resistance genes detection, and whole genome sequencing were performed. Twenty watermelon samples (19%) were contaminated with Salmonella, but none were found in the soil or irrigation water. Among the 20 Salmonella isolates, 2 serovars were identified (Salmonella Liverpool and Salmonella Anatum), belonging to sequence types ST1959 and ST64, respectively. Ten Salmonella isolates showed significant resistance to nalidixic acid, ofloxacin, and ciprofloxacin but were susceptible to all other antibiotics. The coexistence of point mutations (parC:p.T57S) in Quinolone Resistance-Determining Regions and the qnrB19 gene may contribute to quinolone resistance. The study identified 164 virulence genes in the Salmonella isolates. Our study found Salmonella in fresh watermelon during the preharvest season in Bejaia, Algeria. Our study indicates a relatively high prevalence of Salmonella on watermelon samples before harvest. Although we cannot directly compare our results with previous studies, it is crucial to recognize that the absence of comprehensive comparative data underscores the need for further research and surveillance.

An upgraded version of carbapenem inactivation method to detect Bacteroides fragilis carbapenemase.

An increase of carbapenemase-producing Bacteroides fragilis infections is observed. To detect such a resistance in B. fragilis, several tests exist that are expensive or show poor sensitivity and specificity. Therefore, we upgraded the Anaerobic Carbapenem Inactivation Method (Ana-CIM) to easily screen for carbapenemase-producing B. fragilis. The presence of carbapenemase cfiA gene was identified in 50 B. fragilis isolates by PCR. We modified the Ana-CIM by (1) increasing the bacterial inoculum, and (2) measuring the differences in diameter between the negative control and the testing disc. We correctly classified the cfiA-negative and positive isolates and could define a cut-off of positivity at 2 mm. Our modified Ana-CIM allowed to correctly discriminate the 31 cfiA-positive with meropenem MICs ranging from 1 to > 32 µg/mL. We anticipate that our modified Ana-CIM could be used in most clinical laboratories to easily screen for carbapenemase-producing B. fragilis, even at low levels.

Comprehensive Genomic Characterization of Antibiotic Resistance, Virulence, and Clonality in Salmonella Isolates from Wild Animals in Algeria.

This study investigated Salmonella spp. in wild animals in Algeria, focusing on their prevalence, serotypes, antibiotic resistance, and virulence profiles. From fecal samples collected between May 2021 and June 2022, 1.9% showed Salmonella shedding. The identified serotypes included S. Bredeney, S. Enteritidis, S. Altona, and S. Virchow. Except for S. Altona, all isolates were resistant to quinolones, with S. Bredeney strains, exhibiting multidrug resistance. Whole-genome sequencing revealed various resistance genes and mutations in gyrA or parC genes. Additionally, plasmids IncX1 and ColpVC were detected in several isolates. A comprehensive analysis identified 201 virulence genes. These findings contribute to understanding Salmonella in wild animal populations and their potential impact on public health.

A qnrD-Plasmid Promotes Biofilm Formation and Class 1 Integron Gene Cassette Rearrangements in Escherichia coli.

Bacteria within biofilms may be exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. Cell-to-cell contact within biofilms facilitates horizontal gene transfers and favors induction of the SOS response. Altogether, it participates in the emergence of antibiotic resistance. Aminoglycosides at sub-MICs can induce the SOS response through NO accumulation in E. coli carrying the small plasmid with the quinolone resistance qnrD gene (pDIJ09-518a). In this study, we show that in E. coli pDIJ09-518a, the SOS response triggered by sub-MICs of aminoglycosides has important consequences, promoting genetic rearrangement in class 1 integrons and biofilm formation. We found that the integrase expression was increased in E. coli carrying pDIJ09-518a in the presence of tobramycin, which was not observed for the WT isogenic strain that did not carry the qnrD-plasmid. Moreover, we showed that biofilm production was significantly increased in E. coli WT/pDIJ09-518a compared to the WT strain. However, such a higher production was decreased when the Hmp-NO detoxification pathway was fully functional by overexpressing Hmp. Our results showing that a qnrD-plasmid can promote biofilm formation in E. coli and potentiate the acquisition and spread of resistance determinants for other antibiotics complicate the attempts to counteract antibiotic resistance and prevention of biofilm development even further. We anticipate that our findings emphasize the complex challenges that will impact the decisions about antibiotic stewardship, and other decisions related to retaining antibiotics as effective drugs and the development of new drugs.

A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli.

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli, sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD-plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD. Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide (FAD)-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase (FNR)-type transcription factor, related to an O2-responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli. Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

Extended Bacteria Culture-Based Clustering Identifies a Phenotype Associating Increased Cough and Enterobacterales in Stable Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease characterized by airflow limitation. This chronic respiratory disease represents the third leading cause of death worldwide. Alteration of the airway microbiota has been reported to be associated with exacerbation frequency in COPD, but its role on the symptoms in patients at stable state is still incompletely described. This study aimed to determine whether bacteria isolated in sputum can be associated with the clinical features of COPD patients within stable state. Our study highlights, for the first time, that altered microbiota with Enterobacterales is associated with pejorative clinical symptoms in stable COPD patients. The airway microbiota of 38 patients was analyzed using an extended culture approach and mass spectrometry identification. Cluster analysis by principal coordinate analysis of the bacterial communities showed that the patients could be classified into three distinct clusters in our cohort. The clusters showed no differences in proportions of the phylum, but one of them was associated with a high prevalence of Enterobacterales (71.4% in cluster 1 vs. 0% in cluster 3), loss of microbiota diversity, and higher bacterial load (107 vs. 105 CFU/ml, respectively) and characterized by predominant cough and impact on mental health. These novel findings, supported by further studies, could lead to modifying the processing of COPD sputum in the everyday practice of clinical microbiology laboratories.

First description of IncX3 NDM-5-producing plasmid within Escherichia coli ST448 in Mali.

Carbapenem resistance in Enterobacteriaceae has become an increasingly worrying threat. So far, no epidemiological data regarding NDM-producing enterobacterial isolates has been available on these strains in West Africa. The aim of this study was to seek for carbapenemase-producing Enterobacteriaceae clinical strains isolated in Bamako Teaching Hospital in Mali. Of 50 strains isolated between May 2016 and September 2016, we found a ST448 E. coli harbouring an IncX3 plasmid with bla NDM-5 embedded in the ΔISAba125-ble MBL structure. This study reports the first description of NDM-5 in Mali isolated in an undescribed ST E. coli in West Africa.

Genomic analysis of a multidrug-resistant Klebsiella pneumoniae ST11 strain recovered from Barbary deer (Cervus elaphus barbarus) in Akfadou Forest, Algeria.

OBJECTIVES: The emergence and worldwide spread of carbapenemase-producing Enterobacterales (CPE) is a great public-health concern. This study aimed to screen for the presence of CPE isolates from Barbary deer in Akfadou Forest, Béjaïa (Algeria). METHODS: Faecal samples (n=39) were obtained from Barbary deer in Akfadou Forest between March-June 2018. Whole-genome sequencing (WGS) was performed to characterise one representative strain of Klebsiella pneumoniae. Data analysis was performed using online tools. RESULTS: A total of 13 carbapenem-resistantK. pneumoniae isolates were obtained. The isolates showed an identical antimicrobial resistance pattern and were susceptible to colistin and fosfomycin. WGS analysis revealed the complete resistome of K. pneumoniae strain CF21, including blaNDM-1, blaCTX-M-15, blaSHV-182, blaDHA-1, blaOXA-1, aac(3)-IIa, aac(3)-IId, aac(6')-Ib-cr, rmtC, sul1, qnrB9, fosA, tetA, dfrA14, catA2, catB3 and mphA. Multilocus sequence typing (MLST) analysis assigned this strain to the international clone ST11. Plasmid analysis showed that this K. pneumoniae strain possesses five different plasmids including IncA/C2, IncFIA(HI1), IncFIB(K), IncFII(K) and ColRNAI. CONCLUSION: This study reports a multidrug-resistantK. pneumoniae strain recovered from Barbary deer in Algeria and confirms that wild animals could serve as a reservoir of antimicrobial resistance genes.

Phenotypic and genotypic quinolone resistance in Escherichia coli underlining GyrA83/87 mutations as a target to detect ciprofloxacin resistance.

BACKGROUND: Quinolone resistance (QR) is one component of the MDR emerging in Escherichia coli and is of particular concern given the widespread use of fluoroquinolones. OBJECTIVES: To characterize the QR phenotypes and genotypes in E. coli responsible for bloodstream infections and to propose molecular determinants that could be targeted to predict ciprofloxacin resistance. METHODS: E. coli isolates from blood cultures in three French hospitals were studied for quinolone MICs and characterization of genotypic QR determinants (QRg). RESULTS: Among 507 isolates tested for MICs, 148 (29.2%) were resistant to quinolones based on EUCAST breakpoints and 143 (28.2%) harboured at least one QRg. QRg were mainly mutations in the QRDR (138 isolates, 27.2%), with 55.8% of these isolates carrying at least three QRDR mutations. gyrA mutations predominated (92.8%) followed by parC (61.6%), parE (32.6%) and gyrB (1.4%) mutations. Only 4.7% of the isolates harboured a plasmid-mediated quinolone resistance (PMQR) gene: aac(6')-Ib-cr (60.0%) or qnr (qnrS, qnrB) (32.0%). For the first time in France, we reported the qepA4 allele of the plasmid-encoded efflux pump QepA. Only five isolates carried PMQR without a QRDR mutation. The positive predictive value (PPV) for ciprofloxacin resistance was 100% for any QRg and 99.2% for gyrA mutations specifically. CONCLUSIONS: QR observed in E. coli isolates involved in bloodstream infections is still mainly due to QRDR mutations, especially at codons GyrA83/87, which could be used as a molecular target to rapidly detect resistance.

Copper for the Prevention of Outbreaks of Health Care-Associated Infections in a Long-term Care Facility for Older Adults.

OBJECTIVE: We aimed to study the efficacy of copper as an antimicrobial agent by comparing incidence rates during outbreaks in areas equipped vs not equipped with copper surfaces in a long-term facility for dependent older adults (nursing home). DESIGN: Prospective observational pilot study in a nursing home. SETTING AND PARTICIPANT: All persons resident in the nursing home belonging to Reims University Hospital, from February 1, 2015 to June 30, 2016, were included. METHODS: Incidence rates for health care-related infections during outbreaks occurring during the study period were compared between the wing that was equipped and the wing that was not equipped with copper surfaces. Results are expressed as relative risks (RRs) and 95% confidence intervals (95% CIs). RESULTS: During the study period, 556 residents were included; average age was 85.4 ± 9.2 years, and 76% were women. Four outbreaks occurred during the study period: 1 influenza, 1 keratoconjunctivitis, and 2 gastroenteritis outbreaks. The risk of hand-transmitted health care-associated infection was significantly lower in the area equipped with copper surfaces (RR 0.3, 95% CI 0.1-0.5). CONCLUSIONS AND IMPLICATIONS: In our study, copper was shown to reduce the incidence of hand-transmitted health care-associated infections and could represent a relatively simple measure to help prevent HAIs in nursing homes.

Characterization of Carbapenem-Resistant Enterobacteriaceae Clinical Isolates in Al Thawra University Hospital, Sana'a, Yemen.

Objective: The aim of this study was to investigate the resistance mechanisms of carbapenem-resistant Enterobacteriaceae clinical strains recovered from Al Thawra University Hospital, Sana'a, Yemen. Methods: A total of 27 isolates showing decreased susceptibility to carbapenems were obtained from different clinical specimens in Al Thawra Hospital, Sana'a, Yemen. Strains were identified by Matrix Assisted Laser Desorption Ionization Time-Of-Flight spectroscopy. Susceptibility to antibiotics was determined by the disk diffusion method on Mueller Hinton agar. Carbapenemases-encoding genes, extended-spectrum ß-lactamases (ESBLs), and plasmid-mediated quinolone resistance (PMQR) genes were screened by PCR. Bacterial isolates were typed by multilocus sequence typing (MLST). Results: Carbapenemase genes detection and sequencing showed that 18 (66.7%) isolates were Klebsiella pneumoniae (NDM-1, n = 13; NDM-1 + OXA-48, n = 3; OXA-48, n = 1; OXA-232, n = 1), 6 (22.2%) were Escherichia coli (NDM-5, n = 3; OXA-181, n = 2; OXA-48, n = 1), and 3 (11.1%) were Enterobacter cloacae (NDM-1, n = 1; OXA-181, n = 2). In addition the ESBL gene blaCTX-M-15 was detected in 14 K. pneumoniae and 2 E. coli isolates, and the blaCTX-M-216 was found in 1 E. coli isolate. Fifteen isolates were PMQR positive including qnrB1 (n = 1), qnrS1 (n = 5), qnrS4 (n = 2), and aac-(6')-Ib-cr (n = 7). The MLST typing showed a diversity of sequence type (ST) clones including Escherichia coli ST410 (3), ST448 (2), and ST648; Enterobacter cloacae ST78 and ST270; and Klebsiella pneumoniae ST395 (2), ST309, ST23, ST35, ST1728, ST15, ST231, and ST1428. Conclusion: This study reports the first description of OXA-48-like-producing Enterobacteriaceae and NDM-5 enzymes in E. coli in Yemen.

Difficult Gram staining: a case of endocarditis due to Cardiobacterium hominis and review of the literature.

Cardiobacterium hominis est un bacille à Gram négatif responsable d'endocardites infectieuses, principalement chez les patients atteints de pathologies cardiaques ou porteurs de valves. L'identification de cette bactérie est souvent complexe et peut être la cause d'un diagnostic et d'une prise en charge tardifs, source de complications cardiaques. Cet article présente la prise en charge d'une endocardite infectieuse associée à un sepsis à Cardiobacterium hominis, les difficultés d'identification de cette bactérie, ainsi qu'une revue de la littérature sur les infections dues à cette bactérie.

Occurrence of Carbapenemase-Producing Klebsiella pneumoniae in Bat Guano.

The aim of this study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) isolates from bat guano in Bejaia, Algeria. Guano samples (n = 110) were collected in Aokas's cave, Bejaia, Algeria, between March and May 2016. Samples were plated on MacConkey agar supplemented with ertapenem (0.5 mg/L) and vancomycin (32 mg/L). The isolates were identified and antimicrobial susceptibility was determined using disk diffusion method. Carbapenemase, extended spectrum ß-lactamases, plasmid-mediated AmpC, and plasmid-mediated quinolone resistance genes were studied using PCR and sequencing. Clonal relatedness was studied using multilocus sequence typing (MLST). Two CPE isolates were identified as Klebsiella pneumoniae. PCR and sequencing identified the blaOXA-48 in one K. pneumoniae strain (CS34) and blaKPC-3 in the other strain (CS63). K. pneumoniae CS63 was found to carry blaTEM-1 and aac(6')-Ib genes. The MLST showed that K. pneumoniae CS63 was assigned to ST512, whereas K. pneumoniae CS34 belonged to ST1878. This is the first description of CPE from bats' guano.

Performance of a new in-house medium Carba MTL-broth for the rapid detection of carbapenemase-producing Enterobacteriaceae.

INTRODUCTION: The spread of carbapenemase-producing Enterobacteriaceae (CPE) represents a major public health issue. Methods allowing rapid detection of carbapenemases in developing countries are therefore urgently needed. In the current study, we developed a new in-house medium for the rapid detection of CPE isolates, especially OXA-48 producers. METHODOLOGY: A panel of 144 clinical strains previously characterized was tested on in-house Carba MTL-broth medium using four different concentrations of ertapenem (0.5 to 2 mg/L), and compared to chromID® OXA-48 and chromID® CARBA (BioMérieux) media. RESULTS: Comparative evaluation of the Carba MTL-broth with chromID® OXA-48 and chromID® CARBA showed that chromID® OXA-48 and Carba MTL-broth had the highest sensitivity for detection of OXA-48 producers (93.9% and 100%, respectively) comparatively to chromID® CARBA (21.2%). The chromID® OXA-48 had the highest specificity (100%), as compared to the Carba MTL-broth (65.5%) and chromID® CARBA (84.4%) for the detection of OXA-48 producers. CONCLUSIONS: The in-house Carba MTL-broth developed in this study is sensitive, inexpensive, an easy-to-use phenotypic method for the detection of OXA-48-producing enterobacteria. Given the burden of pan-drug resistance, its implementation in the microbiology laboratory of developing countries could be a useful tool for rapid detection of these bacteria.

Copper Alloy Touch Surfaces in Healthcare Facilities: An Effective Solution to Prevent Bacterial Spreading.

In the healthcare environment, microorganisms' cross-transmission between inanimate surfaces and patients or healthcare workers can lead to healthcare-associated infections. A recent interest has grown to create antimicrobial copper touch surfaces, in order to counteract microbial spread in the healthcare environment. For the first time, five French long-term care facilities were at 50% fitted with copper alloys door handles and handrails. Related to the environmental bacterial contamination, 1400 samples were carried out on copper and control surfaces over three years after copper installation. In addition, some copper door handles were taken from the different facilities, and their specific activity against methicillin-resistant S. aureus (MRSA) was tested in vitro. In comparison to control surfaces, copper door handles and handrails revealed significantly lower contamination levels. This difference was observed in the five long-term care facilities and it persists through the three years of the study. High and extreme levels of bacterial contamination were less frequent on copper surfaces. Although, the antibacterial activity of copper surfaces against MRSA was lowered after three years of regular use, it was still significant as compared to inert control surfaces. Therefore, copper containing surfaces are promising actors in the non-spreading of environmental bacterial contamination in healthcare facilities.

Characterization of quinolone resistance mechanisms in Enterobacteriaceae isolated from companion animals in Europe (ComPath II study).

ComPath is an ongoing European programme dedicated to monitor antibiotic susceptibility of bacterial pathogens from diseased dogs and cats. The objective was to characterize determinants associated with quinolone resistance among 160 enrofloxacin non-wild type strains (100 Escherichia coli, 45 Proteus mirabilis, 14 Klebsiella pneumoniae, 1 Enterobacter cloacae) selected among 843 non-duplicate Enterobacteriaceae isolates collected in 12 European countries (2013-2014). These strains with non-wild type MICs of ≥0.25 mg/L, for P. mirabilis ≥0.5 mg/L, were screened for PMQR determinants (qnr, oqxAB, qepA and aac(6')-Ib-cr), and for QRDR mutations in gyrA, gyrB, parC and parE genes. Among them, 20% (32/160) carried at least one PMQR (18/32 qnrB, qnrS or qnrD, 10/32 aac(6')-Ib-cr and 13/32 oqxAB), and 80% (128/160) no PMQR. qnrB was detected in 3 E. coli, 2 K. pneumoniae and 1 E. cloacae strains; qnrS in 6 E. coli and 1 P. mirabilis and aac(6')-Ib-cr in 4 E. coli, 5 K. pneumoniae and 1 E. cloacae strains. All qnrD1 were detected in P. mirabilis. oqxAB was detected in 12/14 K. pneumoniae and 1 E. cloacae. No qepA genes were detected. From the 32 PMQR-positive strains, 10 showed enrofloxacin MICs ≤2 mg/L and 22 MICs ≥8 mg/L, the latter carrying 1-4 mutations in QRDR. For the 128 non-PMQR strains, 37 showed enrofloxacin MICs ≤2 mg/L with 0-2 QRDR mutations, and 91 MICs ≥4 mg/L carrying 1-4 QRDR mutations. In conclusion, qnr was the major PMQR and qnrD only detected in Proteeae. Mutations in QRDR play a markedly greater role in mediating fluoroquinolone resistance than PMQR.

Detection of different classes of carbapenemases: Adaptation and assessment of a phenotypic method applied to Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and proposal of a new algorithm.

A new phenotypic method for detecting carbapenemases has been adapted (assembling of two MAST® kits, including one that contains faropenem to which a temocillin disk has been added) then assessed using 101 bacterial strains (Enterobacteriaceae with assays on Pseudomonas aeruginosa and Acinetobacter baumannii) including 62 which produce genetically identified carbapenemases. Concerning Carbapenemase-Producing Enterobacteriaceae (CPE), there is 100% sensitivity for Klebsiella pneumoniae carbapenemase (KPC, Ambler class A) and OXA-48 (Ambler class D), and 91% for metallo-beta-lactamase (MBL, Ambler class B), with a 97% sensitivity for all carbapenemases, with a specificity of 100%. The test is also efficient for detecting Pseudomonas aeruginosa carbapenemases (sensitivity between 82 and 100% and 100% specificity). The major innovation is the combined use of faropenem and temocillin for reliable detection (excellent performance with 100% sensitivity and specificity) of OXA-48. This study has led to the development of a new algorithm to detect the different classes of carbapenemases, for first-line diagnosis, by combining this modified MAST® test with immunochromatographic methods and molecular biology techniques.

Clonal dissemination of OXA-48-producing Enterobacter cloacae isolates from companion animals in Algeria.

OBJECTIVES: The aim of this study was to investigate carbapenemase-producing Enterobacteriaceae (CPE) in companion animals. METHODS: Between October 2015 and April 2016, 533 rectal swabs were obtained from healthy and diseased pets in different cities in Algeria. Samples were plated on MacConkey agar supplemented with ertapenem (0.5mg/L). Isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibility testing was performed by the disk diffusion method. Carbapenemase, plasmid-mediated AmpC (pAmpC), extended-spectrum ß-lactamase (ESBL) and plasmid-mediated quinolone resistance genes were characterised by PCR. Plasmids were extracted by the Kieser extraction method and were analysed by PCR-based replicon typing (PBRT). The epidemiological relationship between Enterobacter cloacae isolates was determined by random amplified polymorphic DNA (RAPD) analysis and multilocus sequence typing (MLST). RESULTS: From 533 rectal swabs, 12 Enterobacteriaceae (2.3%), including 2 Escherichia coli, 2 Klebsiella pneumoniae and 8 E. cloacae, were recovered from selection plates. The 12 strains were resistant to amoxicillin/clavulanic acid, ticarcillin, piperacillin/tazobactam and ertapenem. All isolates were susceptible to aminoglycosides, imipenem and extended-spectrum cephalosporins. PCR and sequencing identified the blaOXA-48 gene in all isolates. qnrB1 was identified in all E. cloacae isolates. Plasmid analysis showed that the blaOXA-48 gene was localised on a 7-kb untypeable plasmid. RAPD analysis demonstrated the presence of the same profile pattern in the eight E. cloacae isolates. MLST analysis showed that the E. cloacae isolates belonged to ST527. CONCLUSIONS: This study reports for the first time the presence of CPE in horses and pet birds in the world.