High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Pathogenicity assessment of Arcobacter butzleri isolated from Canadian agricultural surface water.

BACKGROUND: Water is considered a source for the transmission of Arcobacter species to both humans and animals. This study was conducted to assess the prevalence, distribution, and pathogenicity of A. butzleri strains, which can potentially pose health risks to humans and animals. Cultures were isolated from surface waters of a mixed-use but predominately agricultural watershed in eastern Ontario, Canada. The detection of antimicrobial resistance (AMR) and virulence-associated genes (VAGs), as well as enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays were performed on 913 A. butzleri strains isolated from 11 agricultural sampling sites. RESULTS: All strains were resistant to one or more antimicrobial agents, with a high rate of resistance to clindamycin (99%) and chloramphenicol (77%), followed by azithromycin (48%) and nalidixic acid (49%). However, isolates showed a significantly (p < 0.05) high rate of susceptibility to tetracycline (1%), gentamycin (2%), ciprofloxacin (4%), and erythromycin (5%). Of the eight VAGs tested, ciaB, mviN, tlyA, and pldA were detected at high frequency (> 85%) compared to irgA (25%), hecB (19%), hecA (15%), and cj1349 (12%) genes. Co-occurrence analysis showed A. butzleri strains resistant to clindamycin, chloramphenicol, nalidixic acid, and azithromycin were positive for ciaB, tlyA, mviN and pldA VAGs. ERIC-PCR fingerprint analysis revealed high genetic similarity among strains isolated from three sites, and the genotypes were significantly associated with AMR and VAGs results, which highlight their potential environmental ubiquity and potential as pathogenic. CONCLUSIONS: The study results show that agricultural activities likely contribute to the contamination of A. butzleri in surface water. The findings underscore the importance of farm management practices in controlling the potential spread of A. butzleri and its associated health risks to humans and animals through contaminated water.

Characterization and genetic analysis of extensively drug-resistant hospital acquired Pseudomonas aeruginosa isolates.

BACKGROUND: The incidence of hospital-acquired infections in extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) has been increasing worldwide and is frequently associated with an increase in mortality and morbidity rates. The aim of this study was to characterize clinical XDR-PA isolates recovered during six months at three different hospitals in Egypt. RESULTS: Seventy hospital-acquired clinical isolates of P. aeruginosa were classified into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR), according to their antimicrobial resistance profile. In addition, the possession of genes associated with mobile genetic elements and genes encoding antimicrobial resistance determinants among isolates were detected using polymerase chain reaction. As a result, a significant percentage of the isolates (75.7%) were XDR, while 18.5% were MDR, however only 5.7% of the isolates were non-MDR. The phenotypic detection of carbapenemases, extended-spectrum ß-lactamases (ESBLs) and metallo ß-lactamase (MBL) enzymes showed that 73.6% of XDR-PA isolates were carbapenemases producers, whereas 75.5% and 88.7% of XDR-PA isolates produced ESBLs and MBL respectively. In addition, PCR screening showed that oxa gene was the most frequently detected gene of carbapenemases (91.4%), while aac(6')-lb gene was mostly detected (84.3%) among the screened aminoglycosides-resistance genes. Furthermore, the molecular detection of the colistin resistance gene showed that 12.9% of isolates harbored mcr-1 gene. Concerning mobile genetic element markers (intI, traA, tnp513, and merA), intI was the highest detected gene as it was amplified in 67 isolates (95.7%). Finally, phylogenetic and molecular typing of the isolates via ERIC-PCR analysis revealed 10 different ERIC fingerprints. CONCLUSION: The present study revealed a high prevalence of XDR-PA in hospital settings which were resistant to a variety of antibiotics due to several mechanisms. In addition, 98% of the XDR-PA clinical isolates contained at least one gene associated with movable genetic elements, which could have aided the evolution of these XDR-PA strains. To reduce spread of drug resistance, judicious use of antimicrobial agents and strict infection control measures are therefore essential.

Molecular typing of clinical multidrug-resistant Klebsiella pneumoniae isolates.

INTRODUCTION: Klebsiella pneumoniae is an opportunistic pathogen which is an important cause of hospital-acquired and antibiotic resistance infections. Therefore, this study aimed to determine the frequency of resistance to antibiotics, as well as the molecular typing of the associated isolates, and compare multiple-locus VNTR analysis (MLVA) and Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) methods to specify the degree to which distinctions can be separated from each other. METHODS AND MATERIALS: One hundred K. pneumoniae isolates were obtained from different sources of infections from patients admitted to hospitals. Antibiotic susceptibility testing was then performed by applying the Kirby-Bauer disk diffusion method. Typing of K. pneumoniae was done by utilizing MLVA and ERIC-PCR methods. RESULTS: Eighty-six multidrug-resistant (MDR) K. pneumoniae isolates were identified, which resistance to ampicillin, trimethoprim/sulfamethoxazole, and ceftriaxone was the most frequent in the considered isolates (100, 93, and 93%, respectively). A total of 50 different antibiotic susceptibility patterns were observed among the MDR K. pneumonia, with the most frequent pattern being resistance to all antibiotics (12.79%) and resistance to all antibiotics except amikacin (10.47%). The isolates were then divided into 37 different MLVA types and seven clonal complexes were obtained from the minimum spanning tree analysis. Finally, the isolates were assigned to 38 different ERIC types. The discriminatory power of MLVA and ERIC methods also showed a value of 0.958, and 0.974. CONCLUSION: Both PCR-typing methods with phenotypic patterns can be useful for the epidemiological typing of K. pneumoniae isolates with the highest performance in discriminating isolates.

Correlation between antimicrobial resistance, biofilm formation, and virulence determinants in uropathogenic Escherichia coli from Egyptian hospital.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is the main etiological agent behind community-acquired and hospital-acquired urinary tract infections (UTIs), which are among the most prevalent human infections. The management of UPEC infections is becoming increasingly difficult owing to multi-drug resistance, biofilm formation, and the possession of an extensive virulence arsenal. This study aims to characterize UPEC isolates in Tanta, Egypt, with regard to their antimicrobial resistance, phylogenetic profile, biofilm formation, and virulence, as well as the potential associations among these factors. METHODS: One hundred UPEC isolates were obtained from UTI patients in Tanta, Egypt. Antimicrobial susceptibility was assessed using the Kirby-Bauer method. Extended-spectrum ß-lactamases (ESBLs) production was screened using the double disk synergy test and confirmed with PCR. Biofilm formation was evaluated using the microtiter-plate assay and microscopy-based techniques. The phylogenetic groups of the isolates were determined. The hemolytic activity, motility, siderophore production, and serum resistance of the isolates were also evaluated. The clonal relatedness of the isolates was assessed using ERIC-PCR. RESULTS: Isolates displayed elevated resistance to cephalosporins (90-43%), sulfamethoxazole-trimethoprim (63%), and ciprofloxacin (53%). Ninety percent of the isolates were multidrug-resistant (MDR)/ extensively drug-resistant (XDR) and 67% produced ESBLs. Notably, there was an inverse correlation between biofilm formation and antimicrobial resistance, and 31%, 29%, 32%, and 8% of the isolates were strong, moderate, weak, and non-biofilm producers, respectively. Beta-hemolysis, motility, siderophore production, and serum resistance were detected in 64%, 84%, 65%, and 11% of the isolates, respectively. Siderophore production was correlated to resistance to multiple antibiotics, while hemolysis was more prevalent in susceptible isolates and associated with stronger biofilms. Phylogroups B2 and D predominated, with lower resistance and stronger biofilms in group B2. ERIC-PCR revealed considerable diversity among the isolates. CONCLUSION: This research highlights the dissemination of resistance in UPEC in Tanta, Egypt. The evident correlation between biofilm and resistance suggests a resistance cost on bacterial cells; and that isolates with lower resistance may rely on biofilms to enhance their survival. This emphasizes the importance of considering biofilm formation ability during the treatment of UPEC infections to avoid therapeutic failure and/or infection recurrence.

Isolation, Identification, and Characterization of Bacillus cereus Group Bacteria Isolated from the Dairy Farm Environment and Raw Milk in Tunisia.

Members of the Bacillus cereus group are well-known opportunistic foodborne pathogens. In this study, the prevalence, hemolytic activity, antimicrobial resistance profile, virulence factor genes, genetic diversity by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) genotyping, and adhesion potential were investigated in isolates from a Tunisian dairy farm environment and raw milk. A total of 200 samples, including bedding, feces, feed, liquid manure, and raw bovine milk, were examined. Based on PCR test targeting sspE gene, 59 isolates were detected. The prevalence of B. cereus group isolates in bedding, feces, liquid manure, feed, and raw milk was 48%, 37.8%, 20%, 17.1%, and 12.5%, respectively. Out of the tested strains, 81.4% showed ß-hemolytic on blood agar plates. An antimicrobial resistance test against 11 antibiotics showed that more than 50% of the isolates were resistant to ampicillin and novobiocin, while a high sensitivity to other antibiotics tested was observed in most isolates. The distribution of enterotoxigenic genes showed that 8.5% and 67.8% of isolates carried hblABCD and nheABC, respectively. In addition, the detection rate of cytotoxin K (cytk), enterotoxin T (bceT), and ces genes was 72.9%, 64.4%, and 5.1%, respectively. ERIC-PCR fingerprinting genotype analysis allowed discriminating 40 different profiles. The adhesion potential of B. cereus group on stainless steel showed that all isolates were able to adhere at various levels, from 1.5 ± 0.3 to 5.1 ± 0.1 log colony-forming unit (CFU)/cm2 for vegetative cells and from 2.6 ± 0.4 to 5.7 ± 0.3 log CFU/cm2 for spores. An important finding of the study is useful for updating the knowledge of the contamination status of B. cereus group in Tunisia, at the dairy farm level.

Evaluation of molecular typing methods for some scab-causing Streptomyces strains from Turkey.

This study was conducted for identifying phylogenetic relationships between 15 scab-causing Streptomyces species including S. bottropensis, S. europaeiscabiei, S. scabiei, S. stelliscabiei and, other 11 Streptomyces sp. All of the strains were originally isolated from symptomatic potatoes in Erzurum Province, The Eastern Anatolia Region of Turkey. Some morphological and biochemical properties of the strains were defined in our former research. Then, 16 s rRNA regions of them were sequenced. After the sequence data assembly, phylogenetic analyzes were performed. The phylogenetic analyses revealed that the strains are involved in the same major group and, substantially similar to reference strains. Additionally, some subgroup formations were also recorded. Moreover, Repetitive element-based PCR (Rep-PCR), Enterobacterial repetitive intergenic consensus (ERIC-PCR), and BOX-PCR fingerprinting molecular typing methods were used for as molecular typing methods. According to our knowledge, this is the first report on phylogenetic relationships of scab-causing Streptomyces species from Turkey. However, the identification of most pathogenic strains remained at the species level.

"Relationship between antibiotic resistance, biofilm formation, virulence factors and source of origin of Pseudomonas aeruginosa environmental isolates with regard to the presence of metallo-ß-lactamase-encoding genes".

A total of 557 water samples were evaluated and of these, 23 were positive for the presence of Pseudomonas aeruginosa. Approximately 91.7% of them were weak biofilm formers. Only 4 isolates showed antimicrobial resistance. All isolates presented Twitching motility, a positive result for the production of pyocyanin, alkaline protease, and hemolysins. The genotypic tests showed: lasA, (95.6%) lasB (95.6%), exoS (95.6%), exoT (91.3%), toxA (91.3%), akgO (91.3%), plcN (91.3%) aprA (86.9%), phzM (78.3%), and pvdA (60.9%). For genes encoding metallo-beta-lactamase, it was found: blaVIM (56.6%), blaSPM (4.3%), and blaSIM (47.8%). A strong association was found between the metallo-beta-lactamase producing genes, nine genes of virulence factors and the motility (r = 0.6231). The very close clonal profile suggests a probable similarity between the isolates from different cities. Thus, P. aeruginosa can be present in water supplies with variable virulence capacities and can generate a huge concern for human, animal, and environmental health.

Evaluation of phenotypical and genotypical methods for the identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility.

AIMS: Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. METHODS AND RESULTS: From 270 S. maltophilia strains identified by VITEK®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and an antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis. MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia, and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing, two strains were identified as Stenotrophomonas rhizophila, showing false-positive results. The confirmed S. maltophilia strains (n = 37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) showed intermediate resistance to sulfamethoxazole-trimethoprim. CONCLUSION: Matrix-assisted laser desorption lonization-time of flight mass spectrometry (MALDI-TOF MS) was a satisfactory methodology for the identification of S. maltophilia, but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing showed low resolution for Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila. ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia.

Molecular characterizations of antibiotic resistance, biofilm formation, and virulence determinants of Pseudomonas aeruginosa isolated from burn wound infection.

BACKGROUND: Burn injuries result in disruption of the skin barrier against opportunistic infections. Pseudomonas aeruginosa is one of the main infectious agents colonizing burn wounds and making severe infections. Biofilm production and other virulence factors along with antibiotic resistance limit appropriate treatment options and time. MATERIALS AND METHODS: Wound samples were collected from hospitalized burn patients. P. aeruginosa isolates and related virulence factors identified by the standard biochemical and molecular methods. Antibiotic resistance patterns were determined by the disc diffusion method and ß-lactamase genes were detected by polymerase chain reaction (PCR) assay. To determine the genetic relatedness amongst the isolates, enterobacterial repetitive intergenic consensus (ERIC)-PCR was also performed. RESULTS: Forty P. aeruginosa isolates were identified. All of these isolates were biofilm producers. Carbapenem resistance was detected in 40% of the isolates, and blaTEM (37/5%), blaVIM (30%), and blaCTX-M (20%) were the most common ß-lactamase genes. The highest resistance was detected to cefotaxime, ceftazidime, meropenem, imipenem and piperacillin, and 16 (40%) isolates were resistant to these antibiotics. The minimum inhibitory concentrations (MIC) of colistin was lower than 2 µg/mL and no resistance was observed. Isolates were categorized to 17 MDR, 13 mono-drug resistance, and 10 susceptible isolates. High genetic diversity was also observed among the isolates (28 ERIC types) and most carbapenem-resistant isolates were classified into four main types. CONCLUSION: Antibiotic resistance, particularly carbapenem resistance was considerable among the P. aeruginosa isolates colonizing burn wounds. Combining carbapenem resistance with biofilm production and virulence factors would result in severe and difficult-to-treat infections.

Biofilm formation, antibiotic resistance and genotyping of Shiga toxin-producing Escherichia coli isolated from retail chicken meats.

1. The Shiga toxin-producing Escherichia coli (STEC) is a hazardous zoonotic agent for chicken meat consumers. This study determined the serogroups and evaluated the virulence genes, antibiotic resistance, biofilm-forming profiles and genetic relationships of STEC isolates in chicken meat.2. A total of 100 samples belonging to dressed-whole chicken and different parts of the chicken (wing, breast, thigh, drumstick) were collected between September and November 2019 from different retail markets in Kayseri, Türkiye.3. Phenotypic (identification, disc diffusion test, Congo red agar and microtitre plate tests) and molecular tests (identification, serogrouping, virulence factors, biofilm, antibiotic susceptibility, 16S rRNA sequencing and enterobacterial repetitive intergenic consensus-PCR for typing of the isolates) were carried out.4. E. coli was isolated from 35% of the samples and 35% of the samples harboured at least one STEC. Among 35 STEC isolates, 3 (8.5%), 6 (17.1%), 2 (5.7%) and 3 (8.5%) were found to be positive for fliCH2, fliCH8, fliCH11, fliCH19 genes, respectively. Out of 35 STEC positive isolates, 4 (11.4%) were identified as E. coli O157, from which 2 (5.7%) were E. coli O157:H7. E. coli O157 was detected in two (10%), one (5%), one (5%) of the thigh, drumstick and whole chicken samples, respectively.5. Biofilm-forming ability was reported in 33 (94.2%) of 35 E. coli isolates, whilst the biofilm-associated genes detected among 35 STEC isolates included csgA (88.5%), fimH (88.5%), bcsA (85.7%), agn43 (14.2%) and papC (8.5%). The STEC strains showed resistance against ampicillin (88.5%) and erythromycin (88.5%), followed by tetracycline (74.2%) and gentamicin (25.7%). However, the distribution of isolates harbouring blaCMY, ere(A), tet(A) and aac(3)-IV antibiotic resistance genes was found to be 17.1%, 11.4%, 85.7% and 5.7%, respectively.6. ERIC-PCR showed that E. coli strains obtained from different parts and whole of chicken samples had genetic diversities. ERIC-PCR patterns grouped strains of 35 STEC into eight clusters designated A-H, with 73% similarity. Proper hygiene measures and staff training are essential for public health during poultry processing and in retail stores to control STEC.

Prevalence of extended-spectrum ß-lactamases, AmpC, and carbapenemases in Proteus mirabilis clinical isolates.

BACKGROUND: Proteus mirabilis is an opportunistic pathogen, causing a variety of community-acquired and nosocomial illnesses. It poses a potential threat to patients via the production of ß-lactamases, which decrease the efficacy of antimicrobial treatment and impair the management of its pathogenicity. Hence, this study was established to determine the prevalence of extended-spectrum ß-lactamases (ESBLs), AmpC, and carbapenemases of P. mirabilis isolated from various clinical specimens. RESULTS: Proteus mirabilis was identified in 20.7% (58/280) of specimens. ESBL producers were present at a rate of 51.7% (30/58). All AmpC-positive isolates (n = 20) produced ESBLs as well, so 66.7% of ESBL-producing isolates coproduced AmpC enzymes. The modified Hodge test confirmed carbapenemase production in six out of seven imipenem nonsusceptible isolates. Of these, only two (5.7%) isolates were also ESBL-and AmpC-positive. Antibiotic resistance reached the highest level for cotrimoxazole (62.1%, n = 36/58 isolates) and the lowest for imipenem (12.1%, n = 7/58 isolates). The levels of multidrug-resistant (MDR) was 41.4% among the tested isolates. The blaSHV (83.3%), blaAmpC (80%), and blaVIM-1 (50%) were the most detected genes in phenotypically confirmed ESBL-, AmpC-, and carbapenemase-producing isolates, respectively. Besides, more than a half of the tested P. mirabilis strains (53%) coproduced ESBLs and AmpC. Moreover, two isolates coproduced ESBLs and AmpC together with carbapenemases. Furthermore, dendrogram analysis showed great genetic divergence based on the 21 different enterobacterial repetitive intergenic consensus (ERIC) patterns (P1-P21) through the 34 ß-lactamase producers. ERIC analysis distinguished clonal similarities between isolates 21 and 22 in P2 and 9 and 10 in P4, which were isolated from the same clinical source and possessed similar patterns of ß-lactamase-encoding genes. CONCLUSION: Hence, there is an urgent need to monitor hospitalized patients and improve healthcare in order to reduce the incidence of infection and outbreaks of infection with antibiotic-resistant Proteus.

Development of sequence-characterized amplified region (SCAR) markers for accurate and differential identification of multienzyme-producing and non-enzymatic Aspergillus strains of industrial importance.

Aspergillus strains are known to produce multiple enzymes of industrial importance. To screen Aspergillus isolates and select a strain with the ability to produce multiple enzymes and discriminate it from non-enzymatic strains, a rapid and accurate approach is required. With this background, a DNA fingerprinting-based study was conducted to develop a simple but accurate molecular detection method with the potential to discriminate multienzyme-producing Aspergillus strains from non-enzymatic strains, irrespective of species. To achieve this, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR was employed to derive group-specific Sequence Characterized Amplified Region (SCAR) markers (i.e., markers corresponding to PCR amplicons of known DNA sequence). To this end, both group-specific (multienzyme-producing and non-enzymatic Aspergillus group) SCAR markers were sought by comparing the ERIC fingerprint profiles and used to develop primers for use in specific and differential identification of multienzyme-producing Aspergillus isolates. As an outcome, the two SCAR-PCR formats were developed. One format is for specific identification of multienzyme-producing Aspergillus strains (SCAR-PCR1), and the other for identifying non-enzymatic Aspergillus strains (SCAR-PCR2). Both SCAR-PCRs were able to discriminate between these two contrasting groups. These formats are simple but accurate and rapid compared to the time-consuming and laborious conventional methods. Therefore, they could be efficient as an alternative strategy for the high-throughput screening of industrially important Aspergillus strains.

Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran.

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed ß-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers.

Epidemiologic and molecular characterization of ß-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients.

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of ß-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, ß-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.

Rhizospheric and endophytic Pseudomonas aeruginosa in edible vegetable plants share molecular and metabolic traits with clinical isolates.

AIM: Pseudomonas aeruginosa, a leading opportunistic pathogen causing hospital-acquired infections, is also commonly found in agricultural settings. However, there are minimal attempts to examine the molecular and functional attributes shared by agricultural and clinical strains of P. aeruginosa. This study investigates the presence of P. aeruginosa in edible vegetable plants (including salad vegetables) and analyses the evolutionary and metabolic relatedness of the agricultural and clinical strains. METHODS AND RESULTS: Eighteen rhizospheric and endophytic P. aeruginosa strains were isolated from cucumber, tomato, eggplant, and chili directly from the farms. The identity of these strains was confirmed using biochemical and molecular assays. The genetic and metabolic traits of these plant-associated P. aeruginosa isolates were compared with clinical strains. DNA fingerprinting and 16S rDNA-based phylogenetic analyses revealed that the plant- and human-associated strains are evolutionarily related. Both agricultural and clinical isolates possessed plant-beneficial properties, including mineral solubilization to release essential nutrients (phosphorous, potassium, and zinc), ammonification, and the ability to release extracellular pyocyanin, siderophore, and indole-3 acetic acid. CONCLUSION: These findings suggest that rhizospheric and endophytic P. aeruginosa strains are genetically and functionally analogous to the clinical isolates. In addition, the genotypic and phenotypic traits do not correlate with plant sources or ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reconfirms that edible plants are the potential source for human and animal transmission of P. aeruginosa.

Silver nanoparticle effect on Salmonella enterica isolated from Northern West Egypt food, poultry, and calves.

A total no. of 65 Salmonella enterica isolates recovered from food samples, feces of diarrheic calves, poultry, and hospital patient in large five cities at Northern West Egypt were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. The 65 Salmonella enterica isolates had the invA gene were grouped into 11 Salmonella enterica serovars with dominance of S. Enteritidis and S. Kentucky serovars. Their resistance pattern were characterized by using 18 antibiotics from different classes. Approximately 80% of the isolates were multidrug resistant (MDR). Enterobacterial repetitive intergenic consequences polymerase chain reaction (ERIC-PCR) typing of 7 strains of S. Enteritidis showed 5 clusters with dissimilarity 25%. S. Enteritidis clusters in 2 main groups A and B. Group A have 2 human strain (HE2 and HE3) and one food origin (FE7) with a similarity 99%. Group B divided into B1 (FE2) and B2 (FE3) with a similarity ratio ≥ 93%, while ERIC-PCR analysis of 5 strains of S. Kentucky revealed 4 ERIC types, clustered in 2 main groups A and B with similarity 75%. We studied the effect of silver nanoparticles (Ag-NPs) on 10 antibiotic resistant strains of S. Enteritidis and S. Kentucky. The broth microdilution minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected. Evaluation of the affection using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed different ratios of Ag-NPs and microorganism as well as at different contact time ended finally with morphological alteration of the bacteria. We submitted new method in vivo to explore the activity of nanosilver in chicken. KEY POINTS: • Importance of ERIC-PCR to determine the relatedness between Salmonella isolates. • Effect of silver nanoparticles to confront the antibacterial resistance. • Studying the effect of silver nanoparticles in vivo on infected chicken with Salmonella.

Characterization of virulence determinants and phylogenetic background of multiple and extensively drug resistant Escherichia coli isolated from different clinical sources in Egypt.

Escherichia coli is a multifaceted microbe since some are commensals, normally inhabiting the gut of both humans and animals while others are pathogenic responsible for a wide range of intestinal and extra-intestinal infections. It is one of the leading causes of septicemia, neonatal meningitis, urinary tract infections (UTIs), cystitis, pyelonephritis, and traveler's diarrhea. The present study aims to survey the distribution and unravel the association of phylotypes, virulence determinants, and antimicrobial resistance of E. coli isolated from different clinical sources in Mansoura hospitals, Egypt. One hundred and fifty E. coli isolates were collected from different clinical sources. Antimicrobial resistance profile, virulence determinants, and virulence encoding genes were detected. Moreover, phylogenetic and molecular typing using ERIC-PCR analysis was performed. Our results have revealed that phylogroup B2 (26.67%) with the greatest content in virulence traits was the most prevalent phylogenetic group. Different virulence profiles and varying incidence of virulence determinants were detected among tested isolates. High rates of resistance to different categories of antimicrobial agents, dramatic increase of MDR (92.67%), and emergence of XDR (4%) were detected. ERIC-PCR analysis revealed great diversity among tested isolates. There was no clustering of isolates according to resistance, virulence patterns, or phylotypes. Our research has demonstrated significant phylogenetic diversity of E. coli isolated from different clinical sources in Mansoura hospitals, Dakahlia governorate, Egypt. E. coli isolates are equipped with various virulence factors which contribute to their pathogenesis in human. The elevated rates of antimicrobial resistance and emergence of MDR and XDR mirror the trend detected globally in recent years. KEY POINTS: • Clinical E. coli isolates exhibited substantial molecular and phylogenetic diversity. • Elevated rates of antimicrobial resistance and emergence of XDR in pathogenic E. coli. • B2 Phylogroup with the highest VS was the most prevalent among pathogenic E. coli.

Molecular Epidemiology and Virulence of Non-Typhoidal Salmonella in Armenia.

In this work, we analysed human isolates of nontyphoidal Salmonella enterica subsp. enterica (NTS), which were collected from salmonellosis cases in Armenia from 1996 to 2019. This disease became a leading food-borne bacterial infection in the region, with the younger age groups especially affected. The isolates were characterised by serotyping, Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) typing, and whole genome sequencing (WGS). The main serotypes were S. Typhimurium, S. Enteritidis, and S. Arizonae. ERIC-PCR indicated a high degree of clonality among S. Typhimurium strains, which were also multidrug-resistant and produced extended spectrum beta-lactamases. During the study period, the frequency of S. Typhimurium and S. Arizonae isolations decreased, but with the increase in S. Enteritidis and other NTS. A total of 42 NTS isolates were subjected to WGS and explored for virulence-related traits and the corresponding genetic elements. Some virulence and genetic factors were shared by all NTS serotypes, while the main differences were attributed to the serotype-specific diversity of virulence genes, SPIs, virulence plasmids, and phages. The results indicated the variability and dynamics in the epidemiology of salmonellosis and a high virulence potential of human NTS isolates circulating in the region.

Antidiarrheal and Antibacterial Activities of Monterey Cypress Phytochemicals: In Vivo and In Vitro Approach.

Monterey cypress (Cupressus macrocarpa) is a decorative plant; however, it possesses various pharmacological activities. Therefore, we explored the phytochemical profile of C. macrocarpa root methanol extract (CRME) for the first time. Moreover, we investigated its antidiarrheal (in vivo), antibacterial, and antibiofilm (in vitro) activities against Salmonella enterica clinical isolates. The LC-ESI-MS/MS analysis of CRME detected the presence of 39 compounds, besides isolation of 2,3,2″,3″-tetrahydro-4'-O-methyl amentoflavone, amentoflavone, and dihydrokaempferol-3-O-α-l-rhamnoside for the first time. Dihydrokaempferol-3-O-α-l-rhamnoside presented the highest antimicrobial activity and the range of values of MICs against S. enterica isolates was from 64 to 256 µg/mL. The antidiarrheal activity of CRME was investigated by induction of diarrhea using castor oil, and exhibited a significant reduction in diarrhea and defecation frequency at all doses, enteropooling (at 400 mg/kg), and gastrointestinal motility (at 200, 400 mg/kg) in mice. The antidiarrheal index of CRME increased in a dose-dependent manner. The effect of CRME on various membrane characters of S. enterica was studied after typing the isolates by ERIC-PCR. Its impact on efflux and its antibiofilm activity were inspected. The biofilm morphology was observed using light and scanning electron microscopes. The effect on efflux activity and biofilm formation was further elucidated using qRT-PCR. A significant increase in inner and outer membrane permeability and a significant decrease in integrity and depolarization (using flow cytometry) were detected with variable percentages. Furthermore, a significant reduction in efflux and biofilm formation was observed. Therefore, CRME could be a promising source for treatment of gastrointestinal tract diseases.