Occurrence, virulence, and resistance genes in Salmonella enterica isolated from an integrated poultry company in Jordan.

Salmonella is considered one of the most common foodborne pathogens worldwide. The annual number of hospitalizations and deaths related to zoonotic salmonellosis, which is transmitted from animals to humans and infects poultry and meat, is expected to be significant. Hence, the primary aims of this research were to isolate and characterize Salmonella species obtained from an integrated poultry company and identify some virulence, and antimicrobial resistance, with a specific concern about colistin resistance genes. A total of 635 samples collected from various sources in an integrated company in Jordan were screened for Salmonella species accompanying their virulence and antimicrobial resistance genes. Samples were collected from parent stock house drag swabs, broiler farms, premix, cecum at the slaughterhouse level, prechilling and postchilling stages, and the final product. Salmonella species were detected in 3% (6/200) of investigated parent stock house drag swabs, 13.8% (11/80) from cloacal swabs from broiler farms, 16.9% (11/65) from boiler farms premix, 24.4% (11/45) from the cecum at slaughterhouse level, 16.4% (9/55) from the prechilling stage, 37.8% (17/45) from the postchilling stage and 53.3% (24/45) from the final product stage. No isolates were detected in feed mills (0/20), parents' premix (0/40), or hatcheries (0/40). Salmonella isolates were resistant to ciprofloxacin (91.0%), nalidixic acid (86.5%), doxycycline (83.1%), tetracycline (83.1%), sulphamethoxazole-trimethoprim (79.8%) and ampicillin (76.4%). Serotyping shows that S. Infantis was the predominant serovar, with 56.2%. Based on the minimum inhibitory concentration (MIC) test, 39.3% (35/89) of the isolates were resistant to colistin; however, no mcr genes were detected. Among antimicrobial-resistant genes, blaTEM was the most prevalent (88.8%). Furthermore, the spvC, ompA, and ompF virulence genes showed the highest percentages (97.8%, 97.8%, and 96.6%, respectively). In conclusion, Salmonella isolates were found at various stages in the integrated company. S. Infantis was the most prevalent serotype. No mcr genes were detected. Cross-contamination between poultry production stages highlights the importance of good hygiene practices. Furthermore, the presence of virulence genes and the patterns of antimicrobial resistance present significant challenges for public health.

Phenotypic and genotypic characterization of Escherichia coli isolated from the chicken liver in relation to slaughterhouse conditions.

Avian pathogenic Escherichia coli (APEC) has been identified as a sub-group of extraintestinal pathogenic E. coli (ExPEC). Recent studies indicate APEC as a potential foodborne zoonotic pathogen and a source or reservoir of human extraintestinal infections. The slaughtering and processing of poultry in low-income countries such as Jordan occurs in two distinct ways: in informal facilities known as Natafat and in formal slaughterhouses. This study compared E. coli phenotypes and genotypes according to slaughtering conditions (formal slaughterhouses vs. informal slaughter facilities). Therefore, liver samples (n = 242) were collected from formal (n = 121) and informal slaughter facilities (n = 121). Results revealed a high prevalence (94.2%) of E. coli among all isolates, with 59 (17 formal and 42 informal) isolates considered avian pathogenic E. coli (APEC) based on the virulence-associated genes. The prevalence of resistance among isolates was relatively high, reaching up to 99% against penicillin and 97% against nalidixic acid. However, the prevalence of resistance was the lowest (1.3%) against both meropenem and imipenem. Based on the MIC test findings, colistin resistance was 46.9% (107/228). The mcr -1 gene prevalence was 51.4% (55/107), of which 17.1 % were from formal plants (6/36) and 68.1% from informal facilities (49/72). Interestingly, only one isolate (0.9%) expressed mcr-10. Escherichia coli O157:H7 and associated virulence genes were found more in informal (n = 15 genes) than in formal slaughterhouses (n = 8). Phylogroups B1, C, and A were the most frequent in 228 E. coli isolates, while G, B2, and clade were the least frequent. In conclusion, these findings highlight the importance of implementing biosecurity measures in slaughterhouses to reduce antibiotic-resistant E. coli spread. Furthermore, this study provides valuable insights into the effects of wet market (Natafat) slaughter conditions on increasing bacterial resistance and virulence.

Risk factors associated with mcr-1 colistin-resistance gene in Escherichia coli broiler samples in northern Jordan.

OBJECTIVES: The purpose of this study was to determine the prevalence of colistin-resistant Escherichia coli carrying mcr-1, and to identify risk factors associated with mcr gene-mediated resistance. METHODS: In total, 385 cloacal samples were collected from 125 broiler farms and a questionnaire containing information about each farm was designed and filled. RESULTS: Most of the antibiotics used in the disk diffusion method were highly resistant in all samples, with tetracycline and penicillin showing 100% and 99.7% resistance, respectively. Additionally, avian pathogenic E. coli (APEC) virulence genes frequency and percentage of APEC were identified, including sitA,iucC, and astA at 77%, 70.5%, and 62% respectively. In total, 214 of 360 isolates were positive for APEC (59.4%). Based on the minimum inhibitory (MIC) test, 58% of the isolates (n = 209 of 360) were resistant to colistin, with 39.7% displaying the mcr-1 gene. The statistical analysis of risk factors that influence colistin resistance prevalence revealed several significant factors, including commercial feed, farm management, sanitization, and antibiotic use. Irregular health checks for workers, non-dipping of feet before entering poultry houses, and the use of commercial poultry feeds all contributed to higher levels of colistin resistance as measured by MIC. On the other hand, doxycycline and commercial feed was 4 and 3.2 times more likely to occur based on the final logistic model of the mcr-1 gene, respectively. CONCLUSION: Our results suggest that better biosecurity protocols should be implemented in poultry farms to reduce antibiotic-resistant bacteria. Additionally, antibiotics should be carefully monitored and used only when necessary.

Multi-drug Resistant Acinetobacter baumannii: Phenotypic and Genotypic Resistance Profiles and the Associated Risk Factors in Teaching Hospital in Jordan.

BACKGROUND: This study aimed to determine the prevalence of Antimicrobial Resistance Genes (ARGs), with a focus on colistin resistance in clinical A. baumannii, as well as the risk factors associated with A. baumannii infection in Jordanian patients. METHODS: In total, 150 A. baumannii isolates were obtained from patients at a teaching hospital. The isolates were tested for antimicrobial susceptibility using disc diffusion and microdilution methods. PCR amplification was used to detect ARGs, and statistical analysis was conducted to evaluate the influence of identified risk factors on the ARGs acquisition. RESULTS: More than 90% of A. baumannii isolates were resistant to monobactam, carbapenem, cephalosporins, Fluoroquinolones, penicillin, and ß-lactam agents. Moreover, 20.6% of the isolates (n = 31) were colistin-resistant. Several ARGs were also detected in A. baumannii isolates. Univariate analysis indicated that risk factors and the carriage of ARGs were significantly associated P ≤ (0.05) with gender, invasive devices, immunodeficiency, systemic diseases, tumors, and covid-19. Logistic regression analysis indicated seven risk factors, and three protective factors were associated with the ARGs (armA, strA, and strB) P ≤ (0.05). In contrast, tetB and TEM were associated with 2 risk factors each P ≤ (0.05). CONCLUSION: Our study indicates a high prevalence of MDR A. baumannii infections in ICU patients, as well as describing the case of colistin-resistant A. baumannii for the first time in Jordan. Additionally, the risk factors associated with ARGs-producing A. baumannii infections among ICU patients suggest a rapid emergence and spread of MDR A. baumannii without adequate surveillance and control measures.

First report of mobile colistin resistance gene mcr-1 in avian pathogenic Escherichia coli isolated from turkeys in the Gaza Strip, Palestine.

Background and Aim: Colistin is used to treat avian pathogenic Escherichia coli (APEC), a microorganism that affects turkey meat production in the Gaza Strip and worldwide. However, the recent emergence of plasmid-borne mobile colistin resistance (mcr) genes in pathogenic E. coli strains is a serious antimicrobial resistance (AMR) challenge for both human and animal health. In December 2018, colistin was banned as a veterinary antimicrobial in the Gaza Strip. This study aimed to detect and track the prevalence of colistin-resistant APEC isolated from turkey flocks in the Gaza Strip. Materials and Methods: This study investigated 239 APEC isolates from turkey flocks in the Gaza Strip between October 2018 and December 2021 (at 6-month intervals). The colistin-resistant APEC strains were detected using the broth microdilution method. The mcr-1 gene was identified using a polymerase chain reaction. Results: The overall colistin resistance among the isolated APECs was 32.2% during the study period. The average resistance in the first interval was 37.5%, which significantly decreased to 9.3% in the last interval. Among the 77 phenotypically resistant isolates, 32.4% were positive for mcr-1. The average abundance of mcr-1 in the first interval was 66.6%, which decreased to 25% in the last interval. Conclusion: To the best of our knowledge, this is the first study reporting the presence of the mcr-1 gene among the APEC isolates from turkeys in the Gaza Strip. Banned veterinary use of colistin significantly decreased the percentage of resistant APEC isolates from turkeys in Gaza Strip. Further studies are needed to investigate other colistin resistance genes and track the emergence of AMR.

Novel spa type and antimicrobial resistance gene profiles of Staphylococcus aureus strains isolated from bovine mastitis in Jordan.

Staphylococcu aureus is the most prevalent microorganism associated with mastitis in cattle. This study was designed to determine the spa types of Staph. aureus and to assess the resistance genes profile of isolated strains in dairy farms in Jordan. In total, 747 milk samples of cattle suffering from subclinical mastitis were collected from 37 dairy farms and tested for Staph. aureus. To detect antimicrobial resistance genes, all 219 strains of Staph. aureus were tested. Furthermore, 21 isolates of Staph. aureus were typed using spa typing. As a result, different proportions of resistance genes were found for Staph. aureus. High resistance genes were in tetK 100%, blaZ 99%, and tetM 97%. Moderate resistance genes were in aac(6')/aph(2'' 52%, ant(4')-Ia 48%, and ermC 41%. Low resistance genes were in ermA is 24%, aph(3')-III is 15%, and mecA is 15%. The spa typing of 21 isolates revealed six spa types, of which five were previously known. For the first time, a novel spa type (t17158) was identified as the main cause of mastitis in dairy cows in Jordan. The identification of resistance genes and spa types is helpful in determining the most effective treatments for cows and plays a significant role in reducing the transmission of pathogens.

Pathological and molecular study of kidneys in apparently healthy cattle and sheep with special reference to Leptospira species in central and northern Jordan.

Background and Aim: Renal pathological conditions can cause significant economic losses for livestock owners. This study investigated and described the gross pathology and histopathology of lesions found in the kidneys of sheep and cattle, with particular attention to the presence of Leptospira spp. Materials and Methods: Three hundred and sixty kidneys from apparently healthy sheep and cattle (208 and 152, respectively) were collected from slaughterhouses in Jordan for gross and histopathological examinations, multiplex polymerase chain reaction (PCR) analysis, and gene sequencing of Leptospira spp. Results: Histopathological analysis of the samples revealed the following conditions: interstitial nephritis (4.44%), glomerulonephritis (4.16%), acute tubular necrosis (17.22%), and renal congestion (4.72%). In addition, in 26.9% of the samples, hyaline material was observed in the interstitium of the cortex and medulla. The PCR results revealed that 83 (23.1%) of the 360 samples were positive for Leptospira spp. 16S rRNA, 42/152 (28%) of cattle, and 41/208 (20%) of sheep. Four samples (two sheep and two cattle) were sequenced and deposited in GenBank with accession numbers from OL701310 to OL701313. Basic Local Alignment Search Tool search and nucleotide similarities between OL701311 and OL701313 resulted in the highest similarities with different Leptospira borgpetersenii strains, whereas OL701310 showed the highest nucleotide similarity (99.2%) with the Leptospira interrogans strain. Similarly, phylogenetic analysis revealed that OL701311 to OL701313 clustered together with different serovars of L. borgpetersenii, whereas OL701310 clustered with the L. interrogans clade. Conclusion: This is the first study to reveal a close association between pathogenic Leptospira spp. and kidney disorders in Jordanian cattle and sheep. These findings may help expand the current understanding of the causes and mechanisms of renal disease in cattle and sheep and contribute to developing more effective prevention and treatment programs.

Emergence of mcr-1 gene and carbapenemase-encoding genes among colistin-resistant Klebsiella pneumoniae clinical isolates in Jordan.

BACKGROUND: Antimicrobial resistance (AMR) is a global threat that requires serious attention, particularly when it is developed against colistin, which is considered one of the 'last-resort' antibiotics for curing an infection. This study aimed to investigate the AMR profile of the Klebsiella (K.) pneumoniae clinical isolates and to obtain the comprehensive characteristics of the carbapenemases among the carbapenem-resistant K. pneumoniae (CR-KP) when isolated. In addition, to detect the colistin resistance and investigate the MCR genes in the clinical K. pneumoniae isolates for the first time in Jordan. METHODS: A total of 179 K. pneumoniae isolates were cultured and they were confirmed using the VITEK 2 system and PCR. The antibiotic susceptibilities, extended-spectrum beta-lactamase (ESßL), multidrug-resistant (MDR), and CR-KP were determined by using the VITEK 2 system, disc diffusion, and the minimum inhibitory concentration (MIC) test. PCR was performed to detect the MCR and carbapenemase genes. RESULTS: The rates of ESßL, MDR, and CR-KP were 48 %, 62 %, and 12.8 %, respectively. High colistin resistance of 49.7 % (89/179) was found. Only one MCR-1 (1.1 %) out of the 89 colistin resistance isolates was detected. Many of the isolates harbored the ESßL genes. In particular, the carbapenem genes were detected in 26 isolates, with 46 % KPC enzyme genes (12/26), 23 % IMP genes (6/26), 19 % OXA-48 genes (5/26), 11.5 % NDM-1 genes (3/26) but no VIM gene was found. The statistical analyses revealed a significant association between colistin resistance and MDR (P ≤ 0.05, Chi-square test). An association between colistin resistance and the Piperacillin, Ceftazidime, Cefpodoxime, Imipenem, Aztreonam, and Tobramycin resistance was noted. CONCLUSION: The study's findings demonstrated the presence of the MCR-1 gene in the K. pneumoniae clinical isolates for the first time in Jordan and indicated that the KPC and IMP encoded carbapenemases were the most prevalent K. pneumoniae carbapenemases in Jordan patients.

Assessment of Immune Response and Efficacy of Essential Oils Application on Controlling Necrotic Enteritis Induced by Clostridium perfringens in Broiler Chickens.

Necrotic enteritis (NE) caused by Clostridium perfringens is one of the most important enteric diseases in poultry. The antibacterial activity of two different essential oil (EO) blends against C. perfringens was investigated both in vitro and in vivo. Additionally, the immunological response to EO treatment was assessed. In the in vitro study, the antibacterial activity of EO formulas and commonly used antibiotics was evaluated against C. perfringens using disk diffusion assay, minimum inhibitory concentration (MIC) assay, and minimum bactericidal concentration (MBC) assay. In the in vivo study, NE experimental infection was performed on 440 Ross broiler chicks at 19 days of age for 4 continuous days. The chicks were treated with either EOs or amoxicillin at 22 days of age for 5 continuous days. One day after the end of treatment, the birds' performance was evaluated by calculating the feed conversion ratio. Serum samples from 120 birds were collected to measure the levels of IL-1ß, IFN-γ, IL-8, IL-10, and IL-17. After that, all birds were slaughtered, and their small intestines were subjected to gross and histopathological evaluation. In addition, bacterial counts in the small intestines were evaluated. In the in vitro study, EOs showed higher antimicrobial activities in comparison with antibiotics against C. perfringens. In the in vivo study, birds treated with EOs showed a significant decrease in bacterial counts, a significant decrease in intestinal lesions, and a significant improvement in performance compared with untreated birds (p < 0.05). Moreover, treating birds with EOs directed the immune system toward an anti-inflammatory pathway. None of the treated birds died due to NE compared with the 10% mortality rate in untreated birds. In conclusion, EOs might be an effective and safe alternative to antibiotics in the treatment of chicken NE.

Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates.

Antimicrobial resistance is a growing global problem that will need a multinational collaborative effort to overcome this serious challenge. The aim of the study is to investigate the potential risk factors associated with the prevalence and distribution of antimicrobial-resistance genes (ARGs) of Escherichia coli isolates obtained from equine fecal samples. One hundred and eighteen horses from different geographical locations and management systems were enrolled in the study and a questionnaire containing information about each individual horse was designed and filled. The enrolled horses belonged to 2 main categories (Hospitalized horses (n = 31), and Non hospitalized horses (n = 87)). In total, 103 E. coli isolates were collected from the 118 horse fecal samples. Genes that are responsible for resistance to ß-lactams, tetracyclines, aminoglycosides, and trimethoprim were detected using uniplex and multiplex polymerase chain reaction (PCR). The prevalence of antimicrobial resistance was significantly higher in hospitalized horses compared to non-hospitalized ones (p ≤ .05), particularly against (trimethoprim, doxycycline, oxytetracycline, and amoxicillin-clavulanic acid). The most prevalent antimicrobial-resistant genes were aminoglycoside resistant genes (strA, strB, and aadA) in percentages; 89%, 85%, and 84%, respectively. Statistical analysis revealed a significant association between risk factors and occurrence of ARGs (p ≤ .05). Significant risk factors include the last treatment and history of antimicrobial administration, breed of horses, use of horses, type of diet fed for horses, practice management and history of last illness. Tetracycline-resistance gene (tetA) was 23 times more likely to be found in the Arabian and local breeds of horses compared to English and warmblood breed. TetA is also 8 times more likely to be found in horses that were fed a natural diet compared to other horses that were fed manufactured/ processed feed. In conclusion, E. coli bacterium can harbor high resistance to different classes of antimicrobials which increases the risk of potential uncontrolled transmission of the multi-drug resistant E. coli bacterium to veterinarians and horse handlers, as well as to the equine population itself.

An overview of colistin resistance, mobilized colistin resistance genes dissemination, global responses, and the alternatives to colistin: A review.

Colistin, also known as polymyxin E, is an antimicrobial agent that is effective against a variety of Gram-negative bacilli, especially the Enterobacteriaceae family. Recently, the wide dissemination of colistin-resistance has brought strong attention to the scientific society because of its importance as the last resort for the treatment of carbapenem-resistant Enterobacteriaceae infections and its possible horizontal transmission. The mobilized colistin resistance (mcr) gene was identified as the gene responsible for unique colistin resistance. Indeed, despite many studies that have revealed a pan variation in the existence of this gene, not only for the mcr genes main group but also for its many subgroups, the problem is growing and worsening day after day. In this regard, this review paper is set to review the updated data that has been published up to the end of 2019 third quarter, especially when related to colistin resistance by the mcr genes. It will include the present status of colistin resistance worldwide, the mcr gene dissemination in different sectors, the discovery of the mcr variants, and the global plan to deal with the threat of antimicrobial resistance. In line with global awareness, and to stop antibiotic misuse and overuse, especially in agricultural animals, the study will further discuss in detail the latest alternatives to colistin use in animals, which may contribute to the elimination of inappropriate antibiotic use and to the help in preventing infections. This review will advance our understanding of colistin resistance, while supporting the efforts toward better stewardship, for the proper usage of antimicrobial drugs in humans, animals, and in the environment.

Potential antimicrobial effect of plant essential oils and virulence genes expression in methicillin-resistant Staphylococcus aureus isolates.

AIM: This study aimed to investigate the antibacterial efficacy of eight commercially available essential oil (EO) blends and characterize the effect on the expression of some virulence genes against methicillin-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS: In vitro evaluation of the antimicrobial effects of oils against MRSA was performed using the disk diffusion method and by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The EOs (A-F) were contained (ß-pinene, carvacrol, carvone, dimethyl trisulfide, linalool, limonene, menthol, monoterpene hydrocarbons, and thymol) in different amounts. In addition, a real-time polymerase chain reaction was also used to determine the gene expression of the virulence genes (intercellular adhesion cluster [ica]-9, ica-15, and RNA III) against MRSA (ATCC 43300) after treatment with selected oils. RESULTS: Among the eight EOs evaluated, EO (D), (E), and (A) showed, in general, the greatest antimicrobial activity against MRSA. EO at 1/3 MIC has effectively down-regulated ica-9 and ica-15 of MRSA by 17.83 and 4.94 folds, respectively. Meanwhile, EO (A) has effectively down-regulated RNAIII by 3.74 folds. Our results indicated that some of the EOs exhibit promising antimicrobial effects against MRSA isolates. Moreover, the results of the analyzed virulence genes related to the pathogenicity of MRSA were down-regulated at the sub-MIC concentrations of EOs, indicated that EOs could be successfully used to suppress the virulence factors and, consequently, decreased the pathogenicity of MRSA. CONCLUSION: These encouraging results indicate that some of the EOs used in this study can be utilized as a natural antibiotic for the treatment of MRSA disease.

Seroprevalence and risk factors of Leptospira serovar Pomona and Leptospira serovar Hardjo infection in dairy cows in Jordan.

INTRODUCTION: This study was carried out to determine the seroprevalence and risk factors associated with Leptospira serovars Pomona and Hardjo infection in dairy cows. METHODOLOGY: Seroprevalence was determined using ELISA using 160 healthy and 80 recently aborted cows. Risk factor assessment was carried out using a pre-validated questionnaire. RESULTS: The true farm seroprevalence of Leptospira serovars Pomona and Hardjo was 92.3% (95% CI: 66%-98%). In healthy cows, the true and apparent cow seroprevalence of Leptospira serovars Pomona and Hardjo were 26.9 (95% CI: 20-34%), 26.25% (95% CI: 20-33%) and 28.75% (95% CI: 22-36%) and 27.5% (95% CI: 21-35%), respectively. Semi-intensive management system (OR = 11.43; P < 0.01), surface water as a source of drinking water (OR = 1.21; P < 0.03), lack of special wear for visitors (OR = 1.39; P < 0.05), and previous history of abortion (OR = 1.02; P < 0.05) were associated with high rate of seropositivity against Leptospira serovars Pomona and Hardjo. In recently aborted cows, the true and apparent seroprevalence rates of Leptospira serovars Pomona and Hardjo were 53.25% (95% CI: 47.5-62%), 53.75% (95% CI: 48.5-63.2%) and 56% (95% CI: 49-61%), 56.25% (95% CI: 49.8-61.2%), respectively. CONCLUSIONS: Leptospirosis is an endemic disease in Jordan and further studies are required to effectively control the disease in dairy cows.

Neutrophil gelatinase-associated lipocalin (NGAL) and insulin-like growth factor (IGF)-1 association with a Mannheimia haemolytica infection in sheep.

This study was aimed at mapping the tissue distribution of some inflammatory parameters associated with a Mannheimia haemolytica (M. haemolytica) infection in sheep. The M. haemolytica was isolated and characterized from the affected lungs of slaughtered animals. Cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, insulin-like growth factor (IGF)-1, as well as the acute-phase protein, neutrophil gelatinase-associated lipocalin (NGAL), were identified in the lung tissues, the serum, and the lymph nodes of M. haemolytica infected sheep, by enzyme-linked immunosorbent assay (ELISA). NGAL and IGF-1 pointed to an innate immune response, and epithelial cell repairing, respectively. The adaptive immune response was identified through the type of cytokines present in the affected sheep, as TNF-α represents the pro-inflammatory cytokines, and IL-10 represents the anti-inflammatory cytokines. M. haemolytica isolates were confirmed by polymerase chain reaction (PCR) and DNA sequences. There was a significant difference in the concentrations of NGAL, IGF-1, TNF-α, and IL-10, as observed in the affected sheep when compared to the healthy sheep. This study, for the first time, closely describes the distribution of some key and new inflammatory parameters in the tissue homogenate of affected lungs.