Changes of composition and antibiotic resistance of fecal coliform bacteria in municipal wastewater treatment plant.

The dynamics of the composition and antibiotic resistance of the fecal coliform bacteria (FCB) in a typical wastewater treatment plant (WWTP) were investigated concerning the seasonal changes. Results showed that WWTP could remove the FCB concentration by 3∼5 logs within the effluent of 104∼105 CFU/L, but the antibiotic resistant rate of FCB species increased significantly after WWTP. The dominant FCB changed from Escherichia coli in the influent (∼73.0%) to Klebsiella pneumoniae in the effluent (∼53.3%) after WWTP, where the Escherichia coli was removed the most, while Klebsiella pneumoniae was the most persistent. The secondary tank removed the most of FCB (by 3∼4 logs) compared to other processes, but increased all the concerned antibiotic resistant rate. The potential super bugs of FCB community showing resistance to all the target antibiotics were selected in the biological treatment unit of WWTP. The FCB showed the highest multiple antibiotic resistance (92.9%) in total which even increased to 100% in the effluent. Klebsiella has the highest antibiotic resistant rate in FCB, with a multiple antibiotic resistance rate of 98.4%. These indicated that the Klebsiella pneumoniae not just Escherichia coli should be specially emphasized after WWTP concerning the health risk associated with FCB community.

Antibiotic resistance profiles of sentinel bacteria isolated from aquaculture in Cambodia.

The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish (Pangasisanodon hypophthalmus) and giant snakehead (Channa micropeltes), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae, Enterobacteriaceae, and Pseudomonadaceae. Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (> 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia.

Vibrio cholerae O1 and Escherichia coli O157:H7 from drinking water and wastewater in Addis Ababa, Ethiopia.

BACKGROUND: In Addis Ababa, Ethiopia, open ditches along innner roads in residential areas serve to convey domestic wastewater and rainwater away from residences. Contamination of drinking water by wastewater through faulty distribution lines could expose households to waterborne illnesses. This prompted the study to assess the microbiological safety of wastewater and drinking water in Addis Ababa, identify the pathogens therein, and determine their antibiotic resistance patterns. RESULTS VIBRIO CHOLERAE: O1, mainly Hikojima serotype, was isolated from 23 wastewater and 16 drinking water samples. Similarly, 19 wastewater and 10 drinking water samples yielded Escherichia coli O157:H7. V. cholerae O1 were 100% resistant to the penicillins (Amoxacillin and Ampicillin), and 51-82% were resistant to the cephalosporins. About 44% of the V. cholerae O1 isolates in this study were Extended Spectrum Beta-Lactamase (ESBL) producers. Moreover, 26% were resistant to Meropenem. Peperacillin/Tazobactam was the only effective ß-lactam antibiotic against V. cholerae O1. V. cholerae O1 isolates showed 37 different patterns of multiple resistance ranging from a minimum of three to a maximum of ten antimicrobials. Of the E. coli O157:H7 isolates, 71% were ESBL producers. About 96% were resistant to Ampicillin. Amikacin and Gentamicin were very effective against E. coli O157:H7 isolates. The isolates from wastewater and drinking water showed multiple antibiotic resistance against three to eight antibiotic drugs. CONCLUSIONS: Open ditches for wastewater conveyance along innner roads in residence areas and underground faulty municipal water distribution lines could be possible sources for V. cholerae O1 and E. coli O157:H7 infections to surrounding households and for dissemination of multiple drug resistance in humans and, potentially, the environment.

Carbapenem-resistant gram-negative bacterial infections and risk factors for acquisition in a Kenyan intensive care unit.

BACKGROUND: Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a critical public health threat globally; however, there are inadequate surveillance data, especially in intensive care units (ICU), to inform infection prevention and control in many resource-constrained settings. Here, we assessed the prevalence of CR-GNB infections and risk factors for acquisition in a Kenyan ICU. METHODS: A hospital-based cross-sectional study design was adopted, recruiting 162 patients clinically presenting with bacterial infection after 48 h of ICU admission, from January to October 2022 at the Nairobi West Hospital, Kenya. Demographics and clinical data were collected by case report form. The type of sample collected, including blood, tracheal aspirate, ascitic tap, urine, stool, and sputum depended on the patient's clinical presentation and were transported to the hospital Microbiology laboratory in a cool box for processing within 2 h. The samples were analyzed by cultured and BD Phoenix system used for isolates' identity and antimicrobial susceptibility. RESULTS: CR-GNB infections prevalence was 25.9% (42/162), with Klebsiella pneumoniae (35.7%, 15/42) and Pseudomonas aeruginosa (26.2%, 11/42) predominating. All isolates were multidrug-resistant (MDR). P. aeruginosa and A. baumannii were 100% colistin-resistant, while K. pneumoniae (33.3%) was tigecycline-resistant. History of antibiotics (aOR = 3.40, p = 0.005) and nasogastric tube (NGT) use (aOR = 5.84, p = < 0.001) were the risk factors for infection. CONCLUSION: Our study highlights high MDR- and CR-GNB infections in ICU, with prior antibiotic exposure and NGT use as risk factors, and diminishing clinical value of colistin and tigecycline. In this study setting and beyond, strict implementation of antimicrobial stewardship programs and adherence to infection prevention and control through monitoring, evaluation and feedback are warranted to curb CR-GNB infections, especially among the risk groups.

Antimicrobial resistance of avian pathogenic Escherichia coli isolated from broiler, layer, and breeder chickens.

Background and Aim: Antimicrobials are extensively used in poultry production for growth promotion as well as for the treatment and control of diseases, including avian pathogenic Escherichia coli (APEC). Poor selection, overuse, and misuse of antimicrobial agents may promote the emergence and dissemination of antimicrobial resistance (AMR) in APEC. This study aimed to assess antimicrobial susceptibility patterns and detect antibiotic resistance genes (ARGs) in APEC isolated from clinical cases of colibacillosis in commercial broiler, layer, and breeder chickens. Materials and Methods: A total of 487 APEC were isolated from 539 across 300 poultry farms in various regions of Nepal. Antimicrobial susceptibility patterns was determined using the Kirby-Bauer disk diffusion and broth microdilution methods. The index of AMR, such as multiple antibiotic resistance (MAR) index, resistance score (R-score), and multidrug resistance (MDR) profile, were determined. Polymerase chain reaction was employed to detect multiple ARGs and correlations between phenotypic and genotypic resistance were analyzed. Results: The prevalence of APEC was 91% (487/539). All of these isolates were found resistant to at least one antimicrobial agent, and 41.7% of the isolates were resistant against 8-9 different antimicrobials. The antibiogram of APEC isolates overall showed the highest resistance against ampicillin (99.4%), whereas the highest intermediate resistance was observed in enrofloxacin (92%). The MAR index and R-score showed significant differences between broiler and layers, as well as between broiler breeder and layers. The number of isolates that were resistant to at least one agent in three or more antimicrobial categories tested was 446 (91.6%) and were classified as MDR-positive isolates. The ARGs were identified in 439 (90.1%) APEC isolates, including the most detected mobilized colistin resistance (mcr1) which was detected in the highest (52.6%) isolates. Overall, resistance gene of beta-lactam (blaTEM), mcr1, resistance gene of sulphonamide (sul1) and resistance gene of tetracycline (tetB) (in broiler), were detected in significantly higher than other tested genes (p < 0.001). When examining the pair-wise correlations, a significant phenotype-phenotype correlation (p < 0.001) was observed between levofloxacin and ciprofloxacin, chloramphenicol and tetracycline with doxycycline. Similarly, a significant phenotype-genotype correlation (p < 0.001) was observed between chloramphenicol and the tetB, and colistin with blaTEM and resistance gene of quinolone (qnrA). Conclusion: In this study, the current state of APEC AMR in commercial chickens is revealed for the first time in Nepal. We deciphered the complex nature of AMR in APEC populations. This information of molecular surveillance is useful to combat AMR in APEC and to contribute to manage APEC associated diseases and develop policies and guidelines to enhance the commercial chicken production.

A Genetic Locus in Elizabethkingia anophelis Associated with Elevated Vancomycin Resistance and Multiple Antibiotic Reduced Susceptibility.

The Gram-negative Elizabethkingia express multiple antibiotic resistance and cause severe opportunistic infections. Vancomycin is commonly used to treat Gram-positive infections and has also been used to treat Elizabethkingia infections, even though Gram-negative organisms possess a vancomycin permeability barrier. Elizabethkingia anophelis appeared relatively vancomycin-susceptible and challenge with this drug led to morphological changes indicating cell lysis. In stark contrast, vancomycin growth challenge revealed that E. anophelis populations refractory to vancomycin emerged. In addition, E. anophelis vancomycin-selected mutants arose at high frequencies and demonstrated elevated vancomycin resistance and reduced susceptibility to other antimicrobials. All mutants possessed a SNP in a gene (vsr1 = vancomycin-susceptibility regulator 1) encoding a PadR family transcriptional regulator located in the putative operon vsr1-ORF551, which is conserved in other Elizabethkingia spp as well. This is the first report linking a padR homologue (vsr1) to antimicrobial resistance in a Gram-negative organism. We provide evidence to support that vsr1 acts as a negative regulator of vsr1-ORF551 and that vsr1-ORF551 upregulation is observed in vancomycin-selected mutants. Vancomycin-selected mutants also demonstrated reduced cell length indicating that cell wall synthesis is affected. ORF551 is a membrane-spanning protein with a small phage shock protein conserved domain. We hypothesize that since vancomycin-resistance is a function of membrane permeability in Gram-negative organisms, it is likely that the antimicrobial resistance mechanism in the vancomycin-selected mutants involves altered drug permeability.

Discovery of a Novel Integrative Conjugative Element ICEAplChn2 Related to SXT/R391 in Actinobacillus pleuropneumoniae.

Objective: The objective of this study was to characterize ICEAplChn2, a novel SXT/R391-related integration and conjugation element (ICE) carrying 19 drug resistance genes, in a clinical isolate of Actinobacillus pleuropneumoniae from swine. Methods: Whole genome sequencing (WGS) of A. pleuropneumoniae CP063424 strain was completed using a combination of third-generation PacBio and second-generation Illumina. The putative ICE was predicted by the online tool ICEfinder. ICEAplChn2 was analyzed by PCR, conjugation experiments, and bioinformatics tools. Results: A. pleuropneumoniae CP063424 strain exhibited high minimum inhibitory concentrations of clindamycin (1,024 mg/L). The WGS data revealed that ICEAplChn2, with a length of 167,870 bp and encoding 151 genes, including multiple antibiotic resistance genes such as erm(42), VanE, LpxC, dfrA1, golS, aadA3, EreA, dfrA32, tetR(C), tet(C), sul2, aph(3)″-lb, aph(6)-l, floR, dfrA, ANT(3″)-IIa, catB11, and VanRE, was found to be related to the SXT/R391 family on the chromosome of A. pleuronipneumoniae CP063424. The circular intermediate of ICEAplChn2 was detected by PCR, but conjugation experiments showed that it was not self-transmissible. Conclusions: To our knowledge, ICEAplChn2 is the longest member with the most resistance genes in the SXT/R391 family. Meanwhile, ATP-binding cassette superfamily was found to be inserted in the ICEAplChn2 and possessed a new insertion region, which is the first description in the SXT/R391 family.

A novel Escherichia coli cell-based bioreporter for quantification of salicylic acid in cosmetics.

Transcription factor-based bioreporters have been extensively studied for monitoring and detecting environmental toxicants. In Escherichia coli, the multiple antibiotic resistance regulator (MarR) induces transcription upon binding to salicylic acid (SA). We generated SA-specific E. coli cell-based bioreporters utilizing the operator region of the mar operon and MarR as components of the reporter and sensing domains, respectively. Although bioreporters based on endogenous MarR and wild-type E. coli cells responded to SA, their sensitivity and selectivity were insufficient for practical sample monitoring. To improve these parameters, we genetically engineered host strains for optimal MarR expression, which enhanced the sensitivity of the biosensor to micromolar quantities of SA with increased selectivity. Under the optimized experimental conditions, the biosensor could quantify SA in environmental samples. For validation, the SA concentration in artificially contaminated SA-containing cosmetic samples was determined using the developed biosensor. Reliability assessment by comparing the concentrations determined using LC-MS/MS revealed > 90% accuracy of the bioreporters. Although bioreporters are not considered standard tools for environmental monitoring, bacterial cell-based bioreporters may serve as alternative tools owing to their affordability and simplicity. The SA biosensor developed in this study can potentially be a valuable tool for monitoring SA in environmental systems. KEY POINTS: • SA-responsive bioreporter is generated by employing mar operon system in E. coli • SA specificity and selectivity were enhanced by genetic/biochemical engineering • The novel bioreporter would be valuable for SA monitoring in environmental systems.

Distribution and antibiotic resistance of vibrio population in an urbanized tropical lake-the Vembanad-in the southwest coast of India.

Among the diverse Vibrio spp. autochthonous to coastal ecosystems, V. cholerae, V. fluvialis, V. vulnificus and V. parahaemolyticus are pathogenic to humans. Increasing sea-surface temperature, sea-level rise and water-related disasters associated with climate change have been shown to influence the proliferation of these bacteria and change their geographic distribution. We investigated the spatio-temporal distribution of Vibrio spp. in a tropical lake for 1 year at a 20-day interval. The abundance of Vibrio spp. was much higher during the south-west monsoon in 2018, when the lake experienced a once-in-a-century flood. The distribution of Vibrio spp. was influenced by salinity (r = 0.3, p < 0.001), phosphate (r = 0.18, p < 0.01) and nitrite (r = 0.16, p < 0.02) in the water. We isolated 470 colonies of Vibrio-like organisms and 341 could be revived further and identified using 16S rRNA gene sequencing. Functional annotations showed that all the 16 Vibrio spp. found in the lake could grow in association with animals. More than 60% of the isolates had multiple antibiotic resistance (MAR) index greater than 0.5. All isolates were resistant to erythromycin and cefepime. The proliferation of multiple antibiotic-resistant Vibrio spp. is a threat to human health. Our observations suggest that the presence of a diverse range of Vibrio spp. is favoured by the low-saline conditions brought about by heavy precipitation. Furthermore, infections caused by contact with Vibrio-contaminated waters may be difficult to cure due to their multiple antibiotic resistances. Therefore, continuous monitoring of bacterial pollution in the lakes is essential, as is the generation of risk maps of vibrio-infested waters to avoid public contact with contaminated waters and associated disease outbreaks.

Antimicrobial resistance and antagonistic features of bivalve-associated Vibrio parahaemolyticus from the south-west coast of India.

Vibrio parahaemolyticus, a potent human and aquatic pathogen, is usually found in estuaries and oceans. Human illness is associated with consuming uncooked/partially cooked contaminated seafood. The study on bivalve-associated V. parahaemolyticus revealed that the post-monsoon season had the highest bacterial abundance (9 ± 1.5 log cfu) compared to the monsoon season (8.03 ± 0.56 log cfu). Antimicrobial resistance (AMR) profiling was performed on 114 V. parahaemolyticus isolates obtained from bivalves. The highest AMR was observed against ampicillin (78%). Chloramphenicol was found to be effective against all the isolates. Multiple antibiotic resistance index values of 0.2 or higher were detected in 18% of the isolates. Molecular analysis of antimicrobial resistant genes (ARGs) revealed the high prevalence (100%) of the TEM-1 gene in the aquatic environment. After plasmid profiling and curing, 41.6% and 100% of the resistant isolates were found to be sensitive to ampicillin and cephalosporins, respectively, indicating the prevalence of plasmid-associated ARGs in the aquatic environment. A study to evaluate the antagonistic properties of Bacillus subtilis, Pseudomonas aeruginosa, and Bacillus amyloliquefaciens against V. parahaemolyticus isolates identified the potential of these bacteria to resist the growth of V. parahaemolyticus.

Microbiota and its antibiotic resistance profile in avocado Guatemalan fruits (Persea nubigena var. guatemalensis) sold at retail markets of Ibarra city, northern Ecuador.

Introduction: Avocados are typically sold in unsanitary conditions at the retail markets in Ecuador, which can raise the risk of microbial contamination. These microorganisms could exhibit multi-antibiotic resistance (MAR), being a serious threat concern to human health. In this study, we aimed to evaluate the microbiota and its antibiotic resistance profile in avocado Guatemalan fruits (Persea nubigena var. guatemalensis), at ripe stage: immature, firm light green (ready to eat in 4 days), peel (AFPE) and pulp (AFPU), and mature intense green (ready to eat) peel (AMPE) and pulp (AMPU), to gain baseline information on the prevalence of MAR bacteria. Methods: Culture-independent (16S rRNA metagenomics) and culture-dependent approach (to detect specific indicator microorganisms) were used. Moreover, antibiotic susceptibility of selected target indicator bacteria was assessed providing information about the antibiotic resistance (AR) among the groups. Results: Based on 16S rRNA gene metagenomic analysis, over 99.78% of reads were classified as bacteria in all samples. Shannon diversity index varies from 1.22 to 2.22, with the highest bacterial population assigned to AFPE samples (1327 species). The highest microbial counts of indicator Staphylococcus spp. (STAPHY), Enterobacter spp. (ENT), and Listeria spp. (LIST), were detected in AMPE samples. Thirty percent of the selected STAPHYs, and 20.91% of Enterobacter (ENT) clones were resistant to various classes of antibiotics. The MAR index varies between 0.25 to 0.88 and was clone-, and fruit ripe stage-dependent. Conclusions: The results indicated that ready to eat avocados contained detectable levels of MAR bacteria, including methicillin resistant (MR)-STAPHY, which may act as a potential vector for the spread of antibiotic resistance. To achieve the increase of the production and marketing of Fuerte cultivar in Ecuador, it is vitally important to consider valuable strategies to protect the fruits at the early ripe stage in future. Thus, it is crucial to set up efficient control measures and develop coordinated strategies to guarantee the microbiological quality of the food.

Resistance Profiles and Virulence Determinants in Biofilm-Forming Enterococcus faecium Isolated from Raw Seafood in Bangladesh.

Pathogenic, antibiotic-resistant, and biofilm-forming bacteria can be transferred to humans through the consumption of contaminated seafood. The present study was carried out to determine antibiotic resistance profiles and virulence determinants in biofilm-forming Enterococcus faecium isolated from seafood in Bangladesh. A total of 150 seafood samples, including shrimp (n = 50), crabs (n = 25), and marine fish (n = 75), were screened using cultural, staining, biochemical, polymerase chain reaction (PCR), Congo red (CR), and disk diffusion (DD) assays. In PCR, E. faecium was detected in 27.3% (41/150; CI95% 20.8; 34.9) of samples, where marine fish (34.7%, CI95% 24.9; 45.9) had the highest prevalence (p < 0.05) compared to crabs (32%, CI95% 17.2; 51.6) and shrimp (14%, CI95% 7.0; 26.1). Thirty-two (78.1%, CI95% 63.3; 88.0) of the E. faecium isolates were determined to be biofilm formers in the CR test, where 43.9% (18/41, CI95% 29.9; 59.0) and 34.2% (14/41, CI95% 21.6; 49.5) of the isolates were strong and intermediate biofilm formers, respectively. In PCR, virulence genes, i.e., pil (100%), ace (92.7%), agg (68.3%), fsrA (65.9%), gelE (63.4%), sprE (53.7%), fsrB (51.2%), and fsrC (43.9%), were detected in E. faecium isolates. All the E. faecium isolates were phenotypically resistant to ≥3 antimicrobial categories and ≥3 antibiotics, including WHO-classified reserve antibiotics linezolid (70.7%) and fosfomycin (19.5%). Moreover, the multiple antibiotic resistance index ranged up to 0.8, showing resistance to ten antibiotics and eight antibiotic classes. In this study, the prevalence of virulence genes and antibiotic resistance was significantly greater (p < 0.05) in strong biofilm-forming E. faecium strains as compared to strains with intermediate and non-biofilm-forming abilities. As far as we know, this study, for the first time in Bangladesh, determined antibiotic resistance and detected virulence genes in biofilm-forming E. faecium isolated from seafood samples. The data from this study could play a significant role in evaluating potential health hazards linked to the ingestion of uncooked or minimally processed seafood.

Phenotypic and molecular characterization of Staphylococcus aureus from mobile phones in Nigeria.

The presence of Staphylococcus aureus, a normal human flora on cellphones of different professionals in Ile-Ife was investigated with a view to determining their antibiotic susceptibility profile and nature of resistance and virulence genes. One hundred swab samples were collected aseptically from mobile phones of various users based on their profession. Surfaces of the mobile phones were swabbed and the streak plate method was used to isolate colonies showing characteristic golden yellow on mannitol salt agar plates. These isolates were further identified using standard microbiological methods. The antibiotic susceptibility of the isolates was determined using Kirby-Bauer's disk diffusion technique. Molecular detection of nuc, mecA and pvl genes in some isolates was carried out by polymerase chain reaction technique. All the 36 isolates obtained in this study were 100% resistant to amoxicillin and augmentin; the isolates also displayed 55.6%, 44.4% and 41.7% resistance to ceftriazone, erythromycin and chloramphenicol, respectively. Based on resistance to oxacillin, prevalence of methicillin resistant Staphylococcus aureus (MRSA) was 11.1%. Only one S. aureus was positive for plasmid analysis. MecA gene was genetically confirmed in four (4) out of the 16 suspected phenotypic MRSA strains, nuc gene was confirmed in all 28 isolates investigated, while there was no pvl gene in the strains investigated. Mobile phones harbor multiple antibiotics resistant S. aureus, which are responsible for important diseases in humans and could be difficult to manage with antibiotics thereby posing serious health risks.

Detection of multidrug-resistant Shiga toxin-producing Escherichia coli in some food products and cattle faeces in Al-Sharkia, Egypt: one health menace.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates. RESULTS: There was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia-Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples (one from H, and 9 from CF) were identified as suspicious E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates (all from CF) were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance (90%, 70%, 60%,60%, and 40%) against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates (CF8, and CF13), both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively. CONCLUSION: The results of this study provides evidence for the occurrence of E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains.

Occurrence and Multidrug Resistance in Strains of Listeria monocytogenes Recovered from the Anaerobic Co-Digestion Sludge Contained in a Single Stage Steel Biodigester: Implications for Antimicrobial Stewardship.

L. monocytogenes is a zoonotic foodborne pathogen with inherent adaptability to tolerate environmental and physiological stresses, thereby causing severe disease outbreaks. Antibiotic resistant foodborne pathogens are a challenge to the food industry. A total of 18 samples were pooled from a bio-digester co-digesting swine manure/pinewood sawdust, and evaluated for the occurrence of bacterium plus total viable counts using the spread plate method. The recovered bacterial isolates were presumptively identified by growth on selective medium and confirmed by biochemical characterisation, leading to the isolation of 43 L. monocytogenes. The isolates were characterized based on their susceptibility to antibiotics via the Kirby-Bauer disc diffusion technique against a panel of 14 antibiotics. Equally, the multiple antibiotic resistance (MAR) index was calculated, and MAR phenotypes generated. The bacterial counts were between 102 and104 cfu/mL. Complete susceptibility (100%) was demonstrated to ampicillin, gentamicin and sulfamethoxazole, which are the drugs of choice in the treatment of listeriosis. In addition, intermediate sensitivity occurred at 25.58% to cefotaxime, and the highest resistance (51.16%) was exhibited against nalidixic acid. The MAR index ranged from 0 to 0.71. Overall, 41.86% of the Listeria isolates displayed multidrug resistance, with 18 different MAR phenotypes, demonstrating CIP, E, C, TET, AUG, S, CTX, NA, AML, NI as the greatest MAR phenotype. It can be concluded that the isolates yielding MAR > 0.2 originated from the farm, where antibiotics had been in routine use. Therefore, strict monitoring of antibiotics use in the farm is crucial to mitigate further increase in antibiotic resistance amongst these bacterial isolates.

Antibiotic Resistance, Virulence Gene Detection, and Biofilm Formation in Aeromonas spp. Isolated from Fish and Humans in Egypt.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates' profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142-0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals.

Biophysical and Biochemical Characterization of the Binding of the MarR-like Transcriptional Regulator Saro_0803 to the nov1 Promotor and Its Inhibition by Resveratrol.

Saro_0803 is a transcriptional factor modulating the transcription of the stilbene-degrading enzyme gene nov1 in Novosphingobium aromaticivorans DSM 12444. Reportedly, Saro_0803 undergoes resveratrol-mediated dissociation from the nov1 promotor and distinguishes resveratrol from its precursors, p-coumaric acid and trans-cinnamic acid, enabling the transcriptional factor to serve as a biosensor component for regulating resveratrol biosynthesis. However, little is known about the molecular mechanisms underlying the Saro_0803 interactions with either the nov1 promotor gene or resveratrol, which undermines the potential for Saro_0803 to be further modified for improved biosynthetic performance and other applications. Here, we report the discovery of the 22 bp A/T-rich Saro_0803 binding site near the -10 box of the nov1 promotor (named nov1p22bp). As validated by molecular docking-guided mutagenesis and binding affinity assays, the Saro_0803 binding of its target DNA sequence relies on charge-predominating interactions between several typical positively charged residues and nucleic acid. Furthermore, we semi-quantified the influence of resveratrol presence on Saro_0803-nov1p22bp interaction and identified a bilateral hydrophobic pocket within Saro_0803 comprising four aromatic residues that are crucial to maintaining the resveratrol binding capability of the transcriptional factor. Our data are beneficial to understanding saro_0803's structural and functional properties, and could provide theoretical clues for future adaptations of this transcriptional factor.

Prevalence of Plasmid-Associated Tetracycline Resistance Genes in Multidrug-Resistant Escherichia coli Strains Isolated from Environmental, Animal and Human Samples in Panama.

Antimicrobial resistance bacteria are nowadays ubiquitous. Its presence has been reported in almost every type of source, from water for agricultural and recreative use, water distribution pipes, and wastewater, to food, fomites, and clinical samples. Enterobacteriaceae, especially Escherichia coli, are not the exception, showing an increased resistance to several antibiotics, causing a global health and economic burden. Therefore, the monitoring of fecal microbiota is important because it is present in numerous reservoirs where gene transfer between commensal and virulent bacteria can take place, representing a potential source of resistant E. coli. In this work, antibiotic resistance profiles of 150 E. coli isolates from environmental, animal, and human samples, collected in three rural areas in Panama, were analyzed. A total of 116 isolates were resistant to at least one of the nine antibiotics tested. Remarkably, almost 100% of these exhibited resistance to tetracycline. Plasmid-associated tetA and tetB genes were detected in 42.86% of the isolates analyzed, tetA being the most prevalent. These results suggest that tetracycline resistance would be used as a convenient indicator of genetic horizontal transfer within a community.

Prevalence and abundance of antibiotic-resistant genes in culturable bacteria inhabiting a non-polar passu glacier, karakorum mountains range, Pakistan.

Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; blaAmpC 6 (100%), blaVIM-1, blaSHV and blaDHA 5 (100%) each, blaOXA-1 1 (100%), blaCMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for blaKPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in blaNDM-1, blaOXA-1, blaSHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance.

Comparison of Virulence-Factor-Encoding Genes and Genotype Distribution amongst Clinical Pseudomonas aeruginosa Strains.

Pseudomonas aeruginosa is an opportunistic pathogen encoding several virulence factors in its genome, which is well-known for its ability to cause severe and life-threatening infections, particularly among cystic fibrosis patients. The organism is also a major cause of nosocomial infections, mainly affecting patients with immune deficiencies and burn wounds, ventilator-assisted patients, and patients affected by other malignancies. The extensively reported emergence of multidrug-resistant (MDR) P. aeruginosa strains poses additional challenges to the management of infections. The aim of this study was to compare the incidence rates of selected virulence-factor-encoding genes and the genotype distribution amongst clinical multidrug-sensitive (MDS) and MDR P. aeruginosa strains. The study involved 74 MDS and 57 MDR P. aeruginosa strains and the following virulence-factor-encoding genes: lasB, plC H, plC N, exoU, nan1, pilA, and pilB. The genotype distribution, with respect to the antimicrobial susceptibility profiles of the strains, was also analyzed. The lasB and plC N genes were present amongst several P. aeruginosa strains, including all the MDR P. aeruginosa, suggesting that their presence might be used as a marker for diagnostic purposes. A wide variety of genotype distributions were observed among the investigated isolates, with the MDS and MDR strains exhibiting, respectively, 18 and 9 distinct profiles. A higher prevalence of genes determining the virulence factors in the MDR strains was observed in this study, but more research is needed on the prevalence and expression levels of these genes in additional MDR strains.