Proteome analysis, genetic characterization, and antibiotic resistance patterns of Klebsiella pneumoniae clinical isolates.

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48) and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021 and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining, and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190 (93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin, 62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone, 53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47% for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant threat to human health as these infections may become difficult to treat.

Using Protein Fingerprinting for Identifying and Discriminating Methicillin Resistant Staphylococcus aureus Isolates from Inpatient and Outpatient Clinics.

In hospitals and other clinical settings, Methicillin-resistant Staphylococcus aureus (MRSA) is a particularly dangerous pathogen that can cause serious or even fatal infections. Thus, the detection and differentiation of MRSA has become an urgent matter in order to provide appropriate treatment and timely intervention in infection control. To ensure this, laboratories must have access to the most up-to-date testing methods and technology available. This study was conducted to determine whether protein fingerprinting technology could be used to identify and distinguish MRSA recovered from both inpatients and outpatients. A total of 326 S. aureus isolates were obtained from 2800 in- and outpatient samples collected from King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia, from October 2018 to March 2021. For the phenotypic identification of 326 probable S. aureus cultures, microscopic analysis, Gram staining, a tube coagulase test, a Staph ID 32 API system, and a Vitek 2 Compact system were used. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), referred to as protein fingerprinting, was performed on each bacterial isolate to determine its proteomic composition. As part of the analysis, Principal Component Analysis (PCA) and a single-peak analysis of MALDI-TOF MS software were also used to distinguish between Methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. According to the results, S. aureus isolates constituted 326 out of 2800 (11.64%) based on the culture technique. The Staph ID 32 API system and Vitek 2 Compact System were able to correctly identify 262 (80.7%) and 281 (86.2%) S. aureus strains, respectively. Based on the Oxacillin Disc Diffusion Method, 197 (62.23%) of 326 isolates of S. aureus exhibited a cefoxitin inhibition zone of less than 21 mm and an oxacillin inhibition zone of less than 10 mm, and were classified as MRSA under Clinical Laboratory Standards Institute guidelines. MALDI-TOF MS was able to correctly identify 100% of all S. aureus isolates with a score value equal to or greater than 2.00. In addition, a close relationship was found between S. aureus isolates and higher peak intensities in the mass ranges of 3990 Da, 4120 Da, and 5850 Da, which were found in MRSA isolates but absent in MSSA isolates. Therefore, protein fingerprinting has the potential to be used in clinical settings to rapidly detect and differentiate MRSA isolates, allowing for more targeted treatments and improved patient outcomes.

The Prevalence of Multidrug-Resistant Acinetobacter baumannii and Its Vaccination Status among Healthcare Providers.

There is growing concern among healthcare providers worldwide regarding the prevalence of multidrug-resistant Acinetobacter baumannii (A. baumannii). Some of the worst hospital-acquired infections, often in intensive care units (ICUs), are caused by this bacterial pathogen. In recent years, the rise in multidrug-resistant A. baumannii has been linked to the overuse of antimicrobial drugs and the lack of adequate infection control measures. Infections caused by this bacterial pathogen are the result of prolonged hospitalization and ICU stays, and they are associated with increased morbidity and mortality. This review outlines the epidemiology, risk factors, and antimicrobial resistance associated with A. baumannii in various countries, with a special focus on the Kingdom of Saudi Arabia. In response to the growing concern regarding this drug-resistant bacteria, fundamental information about its pathology has been incorporated into the development of vaccines. Although these vaccines have been successful in animal models, their effectiveness in humans remains unproven. The review will discuss the development of A. baumannii vaccines, potential related obstacles, and efforts to find an effective strategy against this pathogen.

An Overview of the Public Health Challenges in Diagnosing and Controlling Human Foodborne Pathogens.

Pathogens found in food are believed to be the leading cause of foodborne illnesses; and they are considered a serious problem with global ramifications. During the last few decades, a lot of attention has been paid to determining the microorganisms that cause foodborne illnesses and developing new methods to identify them. Foodborne pathogen identification technologies have evolved rapidly over the last few decades, with the newer technologies focusing on immunoassays, genome-wide approaches, biosensors, and mass spectrometry as the primary methods of identification. Bacteriophages (phages), probiotics and prebiotics were known to have the ability to combat bacterial diseases since the turn of the 20th century. A primary focus of phage use was the development of medical therapies; however, its use quickly expanded to other applications in biotechnology and industry. A similar argument can be made with regards to the food safety industry, as diseases directly endanger the health of customers. Recently, a lot of attention has been paid to bacteriophages, probiotics and prebiotics most likely due to the exhaustion of traditional antibiotics. Reviewing a variety of current quick identification techniques is the purpose of this study. Using these techniques, we are able to quickly identify foodborne pathogenic bacteria, which forms the basis for future research advances. A review of recent studies on the use of phages, probiotics and prebiotics as a means of combating significant foodborne diseases is also presented. Furthermore, we discussed the advantages of using phages as well as the challenges they face, especially given their prevalent application in food safety.

The Development of Diagnostic and Vaccine Strategies for Early Detection and Control of Human Brucellosis, Particularly in Endemic Areas.

Brucellosis is considered one of the most serious zoonotic diseases worldwide. This disease affects both human and animal health, in addition to being one of the most widespread zoonotic illnesses in the Middle East and Northern Africa. Human brucellosis generally presents in a diverse and non-specific manner, making laboratory confirmation of the diagnosis critical to the patient's recovery. A coordinated strategy for diagnosing and controlling brucellosis throughout the Middle East is required, as this disease cannot be known to occur without reliable microbiological, molecular, and epidemiological evidence. Consequently, the current review focuses on the current and emerging microbiological diagnostic tools for the early detection and control of human brucellosis. Laboratory assays such as culturing, serology, and molecular analysis can frequently be used to diagnose brucellosis. Although serological markers and nucleic acid amplification techniques are extremely sensitive, and extensive experience has been gained with these techniques in the laboratory diagnosis of brucellosis, a culture is still considered to be the "gold standard" due to the importance of this aspect of public health and clinical care. In endemic regions, however, serological tests remain the primary method of diagnosis due to their low cost, user-friendliness, and strong ability to provide a negative prediction, so they are commonly used. A nucleic acid amplification assay, which is highly sensitive, specific, and safe, is capable of enabling rapid disease diagnosis. Patients who have reportedly fully healed may continue to have positive molecular test results for a long time. Therefore, cultures and serological methods will continue to be the main tools for diagnosing and following up on human brucellosis for as long as no commercial tests or studies demonstrate adequate interlaboratory reproducibility. As there is no approved vaccine that prevents human brucellosis, vaccination-based control of animal brucellosis has become an important part of the management of human brucellosis. Over the past few decades, several studies have been conducted to develop Brucella vaccines, but the problem of controlling brucellosis in both humans and animals remains challenging. Therefore, this review also aims to present an updated overview of the different types of brucellosis vaccines that are currently available.

Helicobacter pylori Infection: Current Status and Future Prospects on Diagnostic, Therapeutic and Control Challenges.

Helicobacter pylori (H. pylori) infection, which affects approximately half of the world's population, remains a serious public health problem. As H. pylori infection leads to a number of gastric pathologies, including inflammation, gastroduodenal ulcers, and malignancies, early detection and treatment are crucial to preventing the spread of the infection. Multiple extragastric complications, such as iron deficiency anaemia, immune thrombocytopenic purpura, vitamin B12 deficiency, diabetes mellitus, cardiovascular diseases, and certain neurological disorders, have also been linked to H. pylori infection. An awareness of H. pylori and associated health hazards is necessary to minimize or even eradicate the infection. Therefore, there is an urgent need to raise the standards for the currently employed diagnostic, eradication, alternative treatment strategies. In addition, a brief overview of traditional and cutting-edge approaches that have proven effective in identifying and managing H. pylori is needed. Based on the test and laboratory equipment available and patient clinical characteristics, the optimal diagnostic approach requires weighing several factors. The pathophysiology and pathogenic mechanisms of H. pylori should also be studied, focusing more on the infection-causing virulence factors of this bacterium. Accordingly, this review aims to demonstrate the various diagnostic, pathophysiological, therapeutic, and eradication tactics available for H. pylori, emphasizing both their advantages and disadvantages. Invasive methods (such as quick urease testing, biopsy, or culture) or noninvasive methods (such as breath tests, stool investigations, or serological tests) can be used. We also present the most recent worldwide recommendations along with scientific evidence for treating H. pylori. In addition to the current antibiotic regimens, alternative therapies may also be considered. It is imperative to eradicate the infections caused by H. pylori as soon as possible to prevent problems and the development of stomach cancer. In conclusion, significant advances have been made in identifying and treating H. pylori. To improve eradication rates, peptide mass fingerprinting can be used as a diagnostic tool, and vaccines can also eliminate the infection.

The Development of Technology to Prevent, Diagnose, and Manage Antimicrobial Resistance in Healthcare-Associated Infections.

There is a growing risk of antimicrobial resistance (AMR) having an adverse effect on the healthcare system, which results in higher healthcare costs, failed treatments and a higher death rate. A quick diagnostic test that can spot infections resistant to antibiotics is essential for antimicrobial stewardship so physicians and other healthcare professionals can begin treatment as soon as possible. Since the development of antibiotics in the last two decades, traditional, standard antimicrobial treatments have failed to treat healthcare-associated infections (HAIs). These results have led to the development of a variety of cutting-edge alternative methods to combat multidrug-resistant pathogens in healthcare settings. Here, we provide an overview of AMR as well as the technologies being developed to prevent, diagnose, and control healthcare-associated infections (HAIs). As a result of better cleaning and hygiene practices, resistance to bacteria can be reduced, and new, quick, and accurate instruments for diagnosing HAIs must be developed. In addition, we need to explore new therapeutic approaches to combat diseases caused by resistant bacteria. In conclusion, current infection control technologies will be crucial to managing multidrug-resistant infections effectively. As a result of vaccination, antibiotic usage will decrease and new resistance mechanisms will not develop.

How MALDI-TOF Mass Spectrometry Technology Contributes to Microbial Infection Control in Healthcare Settings.

Healthcare settings have been utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) since 2010. MALDI-TOF MS has various benefits over the conventional method of biochemical identification, including ease of use, speed, accuracy, and low cost. This approach can solve many of the obstacles to identifying bacteria, fungi and viruses. As technology advanced, more and more databases kept track of spectra, allowing species with similar morphological, genotypic, and biochemical traits to be identified. Using MALDI-TOF MS for identification has become more accurate and quicker due to advances in sample preparation and database enrichment. Rapid sample detection and colony identification using MALDI-TOF MS have produced promising results. A key application of MALDI-TOF MS is quickly identifying highly virulent and drug-resistant diseases. Here, we present a review of the scientific literature assessing the effectiveness of MALDI-TOF MS for locating clinically relevant pathogenic bacteria, fungi, and viruses. MALDI-TOF MS is a useful strategy for locating clinical pathogens, however, it also has some drawbacks. A small number of spectra in the database and inherent similarities among organisms can make it difficult to distinguish between different species, which can result in misidentifications. The majority of the time additional testing may correct these problems, which happen very seldom. In conclusion, infectious illness diagnosis and clinical care are being revolutionized by the use of MALDI-TOF MS in the clinical microbiology laboratory.

Laboratory Diagnostic Methods and Antibiotic Resistance Patterns of Staphylococcus aureus and Escherichia coli Strains: An Evolving Human Health Challenge.

Raw ground meat is known as a transmission vehicle for biological agents that may be harmful to human health. The objective of the present study was to assess microbiological quality of the ground meats. A total of 280 samples of local and imported chilled meats were randomly collected from retail shops in Buraydah City, Saudi Arabia. The meat samples were microbiologically analyzed using standard methods, peptide mass fingerprinting (PMF) technique, MicroScan Walkaway System (MicroScan) and qPCR System. The imported meat was more bacterially contaminated than local meat, with variable contamination degrees of Staphylococcus aureus (40.33%), Escherichia coli (36.13%), Hafnia alvei (7.56%), Pseudomonas spp. (6.72%), Salmonella spp. (5.88%) and Aeromonas spp. (3.36%). PMF verified all the isolated bacteria by 100%, compared to 75-95% achieved by MicroScan. The gene encoding flagellin (fliC) was recognized in 67.44% of E. coli strains, while the thermonuclease (nuc) and methicillin resistance (mecA) genes were detected in 100% S. aureus and 39.6% of methicillin-resistant S. aureus (MRSA) strains, respectively. The S. aureus and E. coli strains were highly resistant to multiple antibiotics (e.g., ampicillin, amoxicillin-clavulanic acid and cephalothin). For identifying various foodborne pathogens, PMF has been recognized as a powerful and precise analytical method. In light of the increasing use of PMF to detect multidrug-resistant bacteria, this study emphasizes the need for improved ways of treating and preventing pathogens, as well as setting up monitoring systems to guarantee hygiene and safety in meat production.

Brucella species-induced brucellosis: Antimicrobial effects, potential resistance and toxicity of silver and gold nanosized particles.

Brucellosis is an endemic zoonotic disease caused by Brucella species, which are intramacrophage pathogens that make treating this disease challenging. The negative effects of the treatment regime have prompted the development of new antimicrobials against brucellosis. A new treatment modality for antibiotic-resistant microorganisms is the use of nanoparticles (NPs). In this study, we examined the antibacterial activities of silver and gold NPs (SNPs and GNPs, respectively), the resistance developed by Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) strains and the toxicity of both of these NPs in experimental rats. To test the bactericidal effects of the SNPs and GNPs, we used 22 multidrug-resistant Brucella isolates (10 B. melitensis and 12 B. abortus). The minimal inhibitory concentrations (MICs) of both types of NPs were determined utilizing the microdilution technique. To test the stability of resistance, 7 B. melitensis and 6 B. abortus isolates were passaged ten times in culture with subinhibitory concentrations of NPs and another ten times without NPs. Histopathological analysis was completed after rats were given 0.25, 0.5, 1, and 2 mg/kg NPs orally for 28 consecutive days. The MIC values (µg/ml) of the 10-nm SNPs and 20-nm GNPs against B. melitensis were 22.43 ± 2.32 and 13.56 ± 1.22, while these values were 18.77 ± 1.33 and 12.45 ± 1.59 for B. abortus, respectively. After extensive in vitro exposure, most strains showed no resistance to the 10-nm SNPs or 20-nm GNPs. The NPs and antibiotics did not cross-react in any of the evolved Brucella strains. SNPs and GNPs at doses below 2 mg/kg were not harmful to rat tissue according to organ histopathological examinations. However, a greater dose of NPs (2 mg/kg) harmed all of the tissues studied. The bactericidal properties of NPs are demonstrated in this work. Brucella strains develop similar resistance to SNPs and GNPs, and at low dosages, neither SNPs nor GNPs were hazardous to rats.

Pseudomonas species prevalence, protein analysis, and antibiotic resistance: an evolving public health challenge.

Psychrotrophic Pseudomonas is one of the significant microbes that lead to putrefaction in chilled meat. One of the biggest problems in the detection of Pseudomonas is that several species are seemingly identical. Currently, antibiotic resistance is one of the most significant challenges facing the world's health and food security. Therefore, this study was designed to apply an accurate technique for eliminating the identification discrepancy of Pseudomonas species and to study their resistance against various antimicrobials. A total of 320 chicken meat specimens were cultivated, and the isolated bacteria' were phenotypically recognized. Protein analysis was carried out for cultured isolates via Microflex LT. The resistance of Pseudomonas isolates was recorded through Vitek® 2 AST-GN83 cards. Overall, 69 samples were identified as Pseudomonas spp. and included 18 Pseudomonas lundensis (P. lundensis), 16 Pseudomonas fragi (P. fragi), 13 Pseudomonas oryzihabitans (P. oryzihabitans), 10 Pseudomonas stutzeri (P. stutzeri), 5 Pseudomonas fluorescens (P. fluorescens), 4 Pseudomonas putida (P. putida), and 3 Pseudomonas aeruginosa (P. aeruginosa) isolates. Microflex LT identified all Pseudomonas isolates (100%) correctly with a score value ≥ 2.00. PCA positively discriminated the identified isolates into various groups. The antimicrobial resistance levels against Pseudomonas isolates were 81.16% for nitrofurantoin, 71% for ampicillin and ampicillin/sulbactam, 65.22% for cefuroxime and ceftriaxone, 55% for aztreonam, and 49.28% for ciprofloxacin. The susceptibilities were 100% for cefotaxime, 98.55% for ceftazidime, 94.20% for each piperacillin/tazobactam and cefepime, 91.3% for cefazolin. In conclusion, chicken meat was found to be contaminated with different Pseudomonas spp., with high incidence rates of P. lundensis. Microflex LT is a potent tool for distinguishing Pseudomonads at the species level.

Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: An evolution of brucellosis infection control.

Brucellae are intracellular sneaky bacteria and they can elude the host's defensive mechanisms, resulting in therapeutic failure. Therefore, the goal of this investigation was to rapid identification of Brucella species collected from animals and humans in Saudi Arabia, as well as to evaluate their resistance to antibiotics. On selective media, 364 animal samples as well as 70 human blood samples were cultured. Serological and biochemical approaches were initially used to identify a total of 25 probable cultured isolates. The proteomics of Brucella species were identified using the MALDI Biotyper (MBT) system, which was subsequently verified using real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Both Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) were tested for antimicrobial susceptibility using Kirby Bauer method and the E-test. In total, 25 samples were positive for Brucella and included 11 B. melitensis and 14 B. abortus isolates. Twenty-two out of 25 (88%) and 24/25 (96%) of Brucella strains were recognized through the Vitek 2 Compact system. While MBT was magnificently identified 100% of the strains at the species level with a score value more than or equal to 2.00. Trimethoprim-sulfamethoxazole, rifampin, ampicillin-sulbactam, and ampicillin resistance in B. melitensis was 36.36%, 31.82%, 27.27%, and 22.70%, respectively. Rifampin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam resistance was found in 35.71%, 32.14%, 32.14%, and 28.57% of B. abortus isolates, correspondingly. MBT confirmed by microfluidic electrophoresis is a successful approach for identifying Brucella species at the species level. The resistance of B. melitensis and B. abortus to various antibiotics should be investigated in future studies.

Multidrug-resistant Escherichia coli in Raw Milk: Molecular Characterization and the potential impact of camel's Urine as an Antibacterial Agent.

Raw milk is one of the most important vehicles for transmitting various pathogens, especially Escherichia coli (E. coli). Multidrug-resistant pathogens are highly prevalent among mastitic cows in various dairy farms worldwide. Therefore, our current study is based on the identification of E. coli from mastitic cow's milk and their resistance to various antibacterial agents. As well, the impact of camel's urine on multi-drug resistant E. coli were also evaluated. Thirty-three E. coli isolates were recovered from 254 milk samples. All strains were initially identified phenotypically by culturing on specific media and Vitek 2 Compact System. The protein fingerprinting technique was used as a confirmatory method. The Stx1, Stx2 and eae genes were also verified by polymerase chain reaction (PCR). The antimicrobial resistance of E. coli strains was tested by the Vitek 2 AST-GN69 cards. Thirty multi-drug resistant E. coli strains (20 from mastitic milk and 10 from clinical samples) were laboratory tested with different concentrations (100%, 75%, 50% and 25%) of virgin and breeding camel's urine, using the paper disc diffusion method. Our findings showed that 93.94% of E. coli strains were recognized by the Vitek™ 2 system. The results of proteomic investigation illustrated that 100% of E. coli strains were identified at log values ≥2.00. The genotypic identification of the three virulence genes illustrated that 90.1%, 63.64%, and 30.55% of E. coli strains were able to carry the Stx1, eae, and Stx2 genes, respectively. Most strains of E. coli showed strong resistance against cefazolin (78.79%), ceftazidime (66.67%), cefotaxime (60.61%), ceftriaxone (54.55%), and cefepime (39.40%). The results of the antibacterial effect of camel's urine revealed that the mean inhibitory zones of virgin camel's urine were 28 mm, 17 mm, and 14 mm, for the concentrations of 100%, 75%, and 50%, respectively. Whereas; the inhibitory zones for the breeding camel's urine were 18 mm, 0 mm, and 0 mm, for the concentrations of 100%, 75%, and 50%, respectively. We concluded that the majority of E. coli strains were able to harbor some virulence genes and resist many antibiotics. Our study also provided a robust evidence that the camel's urine, particularly from the virgin camels has robust antimicrobial activity against multidrug-resistant E. coli strains.

Acinetobacter baumannii as a community foodborne pathogen: Peptide mass fingerprinting analysis, genotypic of biofilm formation and phenotypic pattern of antimicrobial resistance.

Acinetobacter baumannii (A. baumannii) is one of the most common Gram-negative pathogens that represent a major threat to human life. Because the prevalence of Multidrug-resistant biofilm-forming A. baumannii is increasing all over the world, this may lead to outbreaks of hospital infections. Nonetheless, the role of raw meat as a reservoir for A. baumannii remains unclear. Here our research was aimed to exhibit the frequency, precise identification, and genotyping of biofilm-related genes as well as antimicrobial resistance of A. baumannii isolates of raw meat specimens. Fifty-five A. baumannii strains were recovered from 220 specimens of different animal meat and then identified by Peptide Mass Fingerprinting Technique (PMFT). All identified isolates were genotyped by the qPCR method for the existence of biofilm-related genes (ompA, bap, blaPER-1, csuE, csgA, and fimH). In addition, the antimicrobial resistance against A. baumannii was detected by the Kirby-Bauer method. Based on our findings, the frequency rate of 55 A. baumannii isolates was 46.55%, 32.50%, 15.00%, and 9.68% of sheep, chicken, cow, and camel raw meat samples, respectively. The PMFT was able to identify all strains by 100%. the percentages of csuE, ompA, blaPER-1, bap, and csgA genes in biofilm and non-biofilm producer A. baumannii were 72.73%, 60%, 58.2%, 52.74%, and 25.45%, respectively. In contrast, the fimH was not detected in all non-biofilm and biofilm producer strains. The ompA, bap, blaPER-1, csgA were detected only in biofilm-producing A. baumannii isolates. The maximum degree of resistance was observed against amoxicillin/clavulanic acid (89.10%), gentamicin (74.55%), tetracycline (72.73%), ampicillin (65.45%), and tobramycin (52.73%). In conclusion, our investigation demonstrated the high incidence of multi-drug resistant A. baumannii in raw meat samples, with a high existence of biofilm-related virulence genes of ompA, bap, blaPER-1, csgA. Therefore, it has become necessary to take the control measures to limit the development of A. baumannii.

Enterotoxigenicity and Antibiotic Resistance of Coagulase-Negative Staphylococci Isolated from Raw Buffalo and Cow Milk.

Staphylococcal food poisoning is considered to be one of the most common foodborne illnesses worldwide. Because milk is rich in nutrients and its neutral pH, it leads to the growth of various bacteria. To date, the correlation between enterotoxigenic potential in Staphylococcus species and antimicrobial resistance (AMR), using bioinformatics analysis in buffalo and cow raw milk and the possible health risks from these bacteria, has not been examined in Egypt. A total of 42 Staphylococcus isolates representing 12 coagulase-positive staphylococci (Staphylococcus aureus and Staphylococcus intermedius) and 30 coagulase-negative staphylococci (Staphylococcus capitis, Staphylococcus xylosus, Staphylococcus carnosus, Staphylococcus saccharolyticus, and Staphylococcus auricularis) were isolated. An assay of the antimicrobial resistance phenotypes indicated low resistance against vancomycin (9.5%). The blaZ gene was associated with penicillin G and methicillin resistance and not with sulbactam + ampicillin. The presence of the gene ermB presented the correlation with erythromycin resistance and tetK with tetracycline resistance (correlation index: 0.57 and 0.49, respectively), despite the absence of the same behavior for ermC and tetM, respectively. Interestingly, the gene mecA was not correlated with resistance to methicillin or any other ß-lactam. Correlation showed that slime-producing isolates had more resistance to antibiotics than those of nonslime producers. The multiple correlations between antibiotic resistance phenotypes and resistance genes indicate a complex nature of resistance in Staphylococcus species. The antimicrobial resistance could potentially spread to the community and thus, the resistance of Staphylococcus species to various antibiotics does not depend only on the use of a single antimicrobial, but also extends to other unrelated classes of antimicrobials.

Mutations in VSX2, SOX2, and FOXE3 Identified in Patients with Micro-/Anophthalmia.

Anophthalmia and microphthalmia (A/M) are rare distinct phenotypes that represent a continuum of structural developmental eye defects. Here, we describe three probands from an Egyptian population with various forms of A/M: two patients with bilateral anophthalmia and one with bilateral microphthalmia that were investigated using whole exome sequencing (WES). We identified three causative mutations in three different genes. A new homozygous frameshift mutation c.[422delA];[422delA], p.[N141Ifs∗19];[N141Ifs∗19] in VSX2 was identified in a patient showing bilateral anophthalmia. A previously reported SOX2 deletion c.[70_89del20] p.[N24Rfs∗65];[=] was found in one subject with bilateral anophthalmia. A novel homozygous in-frame mutation c.[431_433delACT];[431_433delACT], p.[Y144del]; [Y144del]) in FOXE3 was identified in a patient with severe bilateral microphthalmia and anterior segment dysgenesis. This study shows that whole exome sequencing (WES) is a reliable and effective strategy for the molecular diagnosis of A/M. Our results expand its allelic heterogeneity and highlight the need for the testing of patient with this developmental anomaly.

Molecular characterization of Escherichia coli isolated from cheese and biocontrol of Shiga toxigenic E. coli with essential oils.

The current research was carried out to study the incidence of Escherichia coli (E. coli) in Egyptian cheese (Kariesh and Ras) and molecular characterization of certain E. coli virulence genes (stx1, stx2, eaeA, hlyA and fimH) using multiplex PCR technique. Biocontrol of E. coli with essential oils (clove and thyme oil) was also studied. A total of 150 random samples of Kariesh and Ras cheese (75 each) were collected from various areas in Governorate of Menoufia. According to our results, the frequency of E. coli isolated from Kariesh and Ras cheese was 16% and 5.3%, respectively. Serological identification classified the E. coli strains into two groups, enterohemorrhagic E. coli (EHEC) serogroup (O26: H11, O91: H21, O111: H2 and O103: H2). While the enterotoxigenic E. coli (ETEC) serogroup were detected as O125: H21 which is the most prevalent strain. O171: H2, O86 and O119: H6 belonging to enteropathogenic E. coli (EPEC). The most prevalent gene detected in E. coli strains was stx1 (87.5%) followed by stx2 (86%), fimH (75%), hlyA (50%) and eaeA (25%) genes. Concerning the antimicrobial activity with essential oils, thyme oil (1%) is considered as the bactericidal effect against E. coli (ATCC35150) with improved the sensory evaluation than clove oil (1%). In conclusion, Kariesh and Ras cheese are extremely tainted with pathogenic E. coli strains, which represent a strong hazard on the human health.

Proteomic characterization and discrimination of Aeromonas species recovered from meat and water samples with a spotlight on the antimicrobial resistance of Aeromonas hydrophila.

Aeromonas is recognized as a human pathogen following ingestion of contaminated food and water. One major problem in Aeromonas identification is that certain species are phenotypically very similar. The antimicrobial resistance is another significant challenge worldwide. We therefore aimed to use mass spectrometry technology for identification and discrimination of Aeromonas species and to screen the antimicrobial resistance of Aeromonas hydrophila (A. hydrophila). A total of 150 chicken meat and water samples were cultured, and then, the isolates were identified biochemically by the Vitek® 2 Compact system. Proteomic identification was performed by MALDI-TOF MS and confirmed by a microchannel fluidics electrophoresis assay. Principal component analysis (PCA) and single-peak analysis created by MALDI were also used to discriminate the Aeromonas species. The antimicrobial resistance of the A. hydrophila isolates was determined by Vitek® 2 AST cards. In total, 43 samples were positive for Aeromonas and comprised 22 A. hydrophila, 12 Aeromonas caviae (A. caviae), and 9 Aeromonas sobria (A. sobria) isolates. Thirty-nine out of 43 (90.69%) Aeromonas isolates were identified by the Vitek® 2 Compact system, whereas 100% of the Aeromonas isolates were correctly identified by MALDI-TOF MS with a score value ≥2.00. PCA successfully separated A. hydrophila, A. caviae and A. sobria isolates into two groups. Single-peak analysis revealed four discriminating peaks that separated A. hydrophila from A. caviae and A. sobria isolates. The resistance of A. hydrophila to antibiotics was 95.46% for ampicillin, 50% for cefotaxime, 45.45% for norfloxacin and pefloxacin, 36.36% for ceftazidime and ciprofloxacin, 31.81% for ofloxacin and 27.27% for nalidixic acid and tobramycin. In conclusion, chicken meat and water were tainted with Aeromonas spp., with a high occurrence of A. hydrophila. MALDI-TOF MS is a powerful technique for characterizing aeromonads at the genus and species levels. Future studies should investigate the resistance of A. hydrophila to various antimicrobial agents.

Antibacterial effects and resistance induction of silver and gold nanoparticles against Staphylococcus aureus-induced mastitis and the potential toxicity in rats.

Staphylococcus aureus (S. aureus) is one of the prevalent mastitis-inducing pathogens worldwide. The resistance of S. aureus to antibiotics is a common issue for dairy farms. Recently, nanoparticles (NPs) have been used for treating antibiotic-resistant bacteria. We therefore aimed to investigate the antimicrobial effect of silver and gold NPs (AgNPs and AuNPs, respectively) and the resistance developed by S. aureus as well as the toxic effects of both NPs in rats. We used 198 S. aureus strains to determine the antibacterial effects of AgNPs and AuNPs. The microdilution method was used to establish the minimum inhibitory concentrations (MICs) of both NPs. To induce resistance, 20 S. aureus strains were passaged 10 times in broth medium with sublethal doses of NPs and an additional 10 times without NPs to examine the stability of resistance. Histopathology was performed after oral administration to the rats with the study doses of 0.25, 0.5, 1, and 2 mg/kg of NPs for 30 days. The MICs of 10-nm AgNPs, 20-nm AgNPs, 10-nm AuNPs, and 20-nm AuNPs against S. aureus were 14.70 ± 1.19 µg/ml, 9.15 ± 0.13 µg/ml, 24.06 ± 2.36 µg/ml, and 18.52 ± 1.26 µg/ml, respectively. Most strains developed strong resistance when treated with 20-nm or 10-nm AgNPs, whereas only two strains were resistant to 10-nm AuNPs and three strains to 20-nm AuNPs. No cross-resistance between NPs and various antibiotics was identified in any of the adapted S. aureus strains. Organ histopathology revealed that 0.25, 0.5, and 1 mg/kg doses of AgNPs and AuNPs were not toxic to rat tissue. In contrast, a higher dose (2 mg/kg) of NPs impaired all organs tested. This study demonstrates the antibacterial effects of NPs. S. aureus strains develop resistance less frequently against AuNPs than AgNPs, and neither AuNPs nor AgNPs was toxic to rats at low doses.

Helicobacter pylori in a poultry slaughterhouse: Prevalence, genotyping and antibiotic resistance pattern.

Although Helicobacter pylori (H. pylori) is a highly significant pathogen, its source remains unclear. Many people consume chicken daily as a source of animal protein worldwide; thus, hygienic methods of supplying chickens for consumption are critical for public health. Therefore, our study examined the distribution of the glmM (ureC), babA2, vacA and cagA virulence genes in H. pylori strains in chicken meat and giblets (gizzards and livers) and the resistance of the strains to various antibiotics. Ninety chicken meat, gizzard and liver samples were obtained from a semi-automatic abattoir in Sadat City, Egypt, and were cultured and preliminarily analyzed using biochemical tests. The presence of the ureC, babA2, vacA and cagA genotypes was tested for in samples positive for H. pylori by multiplex polymerase chain reaction (Multiplex-PCR). The resistance of H. pylori to various antimicrobial drugs was tested using the disc diffusion method. In total, 7 of the 90 chicken samples were positive for H. pylori (7.78%); in 3/7 (42.85%) samples, the bacteria were found in the chicken liver, while the bacteria were found in the meat in 2/7 (28.57%) and in the gizzard in 2/7 (28.57%) samples. The total prevalence of both the ureC and babA2 genes in the isolated H. pylori strains was 100%, while the prevalence of the vacA and cagA genes was 57.1% and 42.9%, respectively. The resistance of H. pylori to the antibiotics utilized in our study was 100% for streptomycin; 85.7% for amoxicillin and penicillin; 71.4% for oxytetracycline, nalidixic acid and ampicillin; 57.1% for sulfamethoxazole and erythromycin; and 42.9% for neomycin, chloramphenicol and norfloxacin. In conclusion, the chicken meat and giblets were tainted by H. pylori, with a higher occurrence of the ureC, babA2, vacA and cagA genotypes. Future investigations should investigate the resistance of H. pylori to various antimicrobial agents in Egypt.