Occurrence of carbapenem-resistant hypervirulent Klebsiella pneumoniae in oysters in Egypt: a significant public health issue.

BACKGROUND: The global dissemination of critical-priority carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) via food sources represents a significant public health concern. Epidemiological data on CR-hvKp in oysters in Egypt is limited. This study aimed to investigate the potential role of oysters sold in Egypt as a source for carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKp), and CR-hvKp and assess associated zoonotic risks. METHODS: A sample of 330 fresh oysters was randomly purchased from various retail fish markets in Egypt and divided into 33 pools. Bacteriological examination and the identification of Klebsiella pneumoniae were performed. Carbapenem resistance in K. pneumoniae isolates was determined by phenotypic and molecular methods. Additionally, the presence of hypervirulent K. pneumoniae was identified based on virulence gene markers (peg-344, rmpA, rmpA2, iucA, and iroB), followed by a string test. The clustering of CR-hvKp strains was carried out using R with the pheatmap package. RESULTS: The overall prevalence of K. pneumoniae was 48.5% (16 out of 33), with 13 isolates displaying carbapenem resistance, one intermediate resistance, and two sensitive. Both carbapenem-resistant K. pneumoniae and carbapenem-intermediate-resistant K. pneumoniae strains exhibited carbapenemase production, predominantly linked to the blaVIM gene (68.8%). HvKp strains were identified at a rate of 62.5% (10/16); notably, peg-344 was the most prevalent gene. Significantly, 10 of the 13 CRKP isolates possessed hypervirulence genes, contributing to the emergence of CR-hvKp. Moreover, cluster analysis revealed the clustering of two CR-hvKp isolates from the same retail fish market. CONCLUSION: This study provides the first insight into the emergence of CR-hvKp among oysters in Egypt. It underscores the potential role of oysters as a source for disseminating CR-hvKp within aquatic ecosystems, presenting a possible threat to public health.

Biofilms and efflux pump regulatory gene (mexR) in multidrug-resistant Pseudomonas aeruginosa isolated from migratory birds in Egypt.

Background and Aim: Multidrug-resistant (MDR) Pseudomonas aeruginosa is a global threat to public health. This study aimed to determine biofilms and efflux pump regulatory gene (mexR) in MDR P. aeruginosa isolates. Materials and Methods: A total of 42 fecal samples of aquatic migratory birds collected during hunting season in Egypt were evaluated for the detection of P. aeruginosa according to standard culture-based methods. The antibiotic susceptibility of P. aeruginosa strains was evaluated using disk diffusion methods. The biofilm formation ability of the isolates was phenotypically determined using a colorimetric microtitration plate assay. Polymerase chain reaction amplification was performed to detect biofilm genes (PelA and PslA) and mexR. Results: In total, 19 isolates (45.2%) were recovered from the 42 fecal samples of migratory birds. All isolates were identified as MDR P. aeruginosa, and 78.9% of the strains produced biofilms at different degrees. Molecular detection of biofilm extracellular polymeric substances revealed that PelA was the most predominant gene in the biofilm-producing isolates, followed by PslA. mexR was detected in 63.2% of MDR P. aeruginosa isolates, and its prevalence was higher in non-biofilm-producing strains (75%) than in biofilm-producing strains (60%). Conclusion: Antibiotic resistance in P. aeruginosa isolates recovered from migratory birds through various mechanisms is a major public and animal health problem. It is important to consider the significance of migratory birds in disease transmission.

Horse: a potential source of Cryptococcus neoformans and Cryptococcus gattii in Egypt.

BACKGROUND: Cryptococcosis is an opportunistic mycozoonosis of global significance in a wide variety of host species. In equines, cryptococcosis is uncommon, and sporadic cases have been reported with rhinitis, sinusitis, pneumonia, and meningitis. Cryptococcus spp. represents a potential risk for immunosuppressed and healthy persons. In Egypt, epidemiological data on cryptococcal infection in horses are limited. The current study was carried out to investigate the occurrence of Cryptococcus spp. in horses and its possible role in the epidemiology of such disease in Egypt. A total of 223 samples was collected from different localities in Egypt included 183 nasal swabs from horses, 28 nasal swabs from humans, and 12 soil samples. Bacteriological examination and the identification of Cryptococcus spp. were performed. Molecular serotyping of Cryptococcus spp. was determined by multiplex PCR using CNa-70S/A-CNb-49S/A. The virulence genes (LAC1, CAP59, and PLB1) of the identified isolates were detected by PCR. Moreover, sequencing and phylogenetic analysis of the C. gattii gene from horses, humans, and soil isolates found nearby were performed. RESULT: The overall occurrence of Cryptococcus spp. in horses were 9.3, 25, and 10.7% in horses, the soil, and humans, respectively. Molecular serotyping of the Cryptococcus spp. isolates recovered from the nasal passages of horses proved that C. gattii (B), C. neoformans, and two hybrids between C. neoformans (A) and C. gattii (B) were identified. Meanwhile, in case of soil samples, the isolates were identified as C. gattii (B). The human isolates were serotyped as C. gattii in two isolates and C. neoformans in only one isolate. Molecular detection of some virulence genes (LAC1), (CAP59), and (PLB1) were identified in both C. gattii and C. neoformans isolates. The C. gattii gene amplicons of the isolates from horses, humans, and the soil were closely related. CONCLUSION: This study provides the first insights into the Egyptian horse ecology of Cryptococcus species and highlights the role of horses as asymptomatic carriers in disseminating the potentially pathogenic Cryptococcus spp. It also presents the possible risk of cryptococcosis infection in humans.

Pathways of Methicillin-Resistant Staphylococcus aureus in Animal Model: New Insights Regarding Public Health.

BACKGROUND: Staphylococcus aureus is considered one of the major threats regarding food safety worldwide. Methicillin-resistant S. aureus (MRSA) strains in livestock, companion animals, and wild animals continue to be a potential risk to people working with them. AIM: The current research aims to investigate the potential pathways of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains in the body after oral infection using the experimental mouse model. METHODS: Seven groups of SPF male mice were purchased and housed. On day 1, six groups of mice were infected orally by the sterile gastric probe using 100 µL/mice of LA-MRSA bacterial suspension (1 × 108 colony-forming units (CFU)/mL). The remaining group was kept as negative controls. Over 15 days, these animals have been monitored. Fresh fecal samples were screened for LA-MRSA at day 0, day 7 and day 14 following oral administration of MRSA strains. All animals were sacrificed at day 15, and internal organs (liver, lung, kidney, and intestine) were harvested aseptically and divided into two sections. The first part was histopathologically investigated, while the other half has been tested for LA-MRSA re-isolation. RESULT: The oral challenge of mice by MRSA strains showed that MRSA was re-isolated from feces and intestines of all inoculated mice groups and from internal organs (liver, lung, kidney and intestine) of most mice. Results were confirmed by the detection of the bacteria in gram-stained tissue sections and changes in H&E-stained histopathological tissue sections from these organs. CONCLUSION: Data from the present study indicate the possible colonization of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in internal organs following oral infection and thus posing a risk for food-borne infection of MRSA. Infected animals could pass LA-MRSA through feces again, resulting in increased dispersion and environmental contamination.

Isolation and characterization of E. coli strains causing intramammary infections from dairy animals and wild birds.

The study was conducted to estimate the prevalence of Escherichia coli (E. coli) in sub-clinically mastitic (SCM) animals, and in wild and migratory birds which may act as reservoir disseminating such pathogen. Farm hygiene, management and milking procedures were listed through a questionnaire. Thirty lactating cows and 15 lactating buffaloes from five small-scale dairy farms were randomly selected and screened for subclinical mastitis (SCM) using California Mastitis Test (CMT) and somatic cell count (SCC). In addition, 80 teat skin swabs, 5 drinking water samples and 38 wild and migratory bird faecal matter were also collected. All samples were processed for E. coli isolation by culturing on Levine's Eosin Methylene Blue (L-EMB) agar, followed by purification and biochemical identification. Positive samples were subjected to molecular identification and serotyping. In addition, the presence of extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing E. coli have been reported by antimicrobial sensitivity testing. Escherichia coli were isolated from 7.7%, 50% and 50% of the positive CMT cows' quarters, cows' composite and buffaloes' composite milk samples, respectively. In addition, 14% of cows' teats, 20% of water samples, 70% of faecal matter from wild bird, and 33.3% of faecal matter from migratory waterfowls were carrying E. coli. Serotyping, antibiotic-resistant pattern and phylogenetic analysis have pointed the bearable implication of milking hygiene and wild birds in disseminating E. coli strains causing intramammary infections.

Evidence of colistin resistance genes (mcr-1 and mcr-2) in wild birds and its public health implication in Egypt.

Background: Antimicrobial resistance has become one of the most severe global threats to human and veterinary Medicine. colistin is an effective therapeutic agent against multi-drug-resistant pathogens. However, the discovery of transferable plasmids that confer resistance to colistin (mcr-1) has led to challenges in medical science. This study describes the role of wild birds in the harbouring and environmental spread of colistin-resistant bacteria, which could pose a potential hazard to human and animal health. Methods: In total, 140 faecal samples from wild birds (migratory and resident birds) were tested. Twenty surface water samples were collected from the area in which wild bird trapping was conducted, and 50 human stool samples were collected from individuals residing near the surface water sources and farm buildings. Isolation and identification of Enterobacteriaceae and Pseudomonas aeruginosa from the different samples were performed using conventional culture techniques and biochemical identification. PCR amplification of the mcr genes was performed in all positive isolates. Sequencing of mcr-1 genes from three randomly selected E. coli carrying mcr-1 isolates; wild birds, water and humans was performed. Result: The bacteriological examination of the samples showing isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and P. aeruginosa. The results of multiplex PCR of the mcr genes revealed that E. coli was the most prevalent gram-negative bacterium harbouring the mcr genes, whereas a low prevalence was observed for K. pneumoniae. The prevalence of mcr-1 in resident birds, migratory birds, water sources and humans were 10.4, 20,16.6 and 9.6% while the prevalence of mcr-2 were 1.4, 3.6, 11.1 and 9.6%, respectively. Sequencing of the mcr-1 gene from the three E. coli carrying mcr-1 isolates indicated a possible correlation between the wild bird and surface water isolates. Conclusion: The detection of mcr-1-positive bacteria in wild birds in Egypt indicates the possible environmental dissemination of this gene through bird activity. The impact of the interaction between domestic and wild animals on public health cannot be overlooked.

Carbapenem-resistant Pseudomonas Aeruginosa Originating from Farm Animals and People in Egypt.

INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has become the leading cause of health care-associated infections. Treatment is difficult due to the lack of an effective antimicrobial therapy, and mortality is high. This study investigated the occurrence of CRPA in farm animals (buffaloes and cattle), livestock drinking water, and humans in Egypt. MATERIAL AND METHODS: A total of 180 samples were examined: 50 faecal each from buffaloes and cattle, 30 of livestock drinking water, and 50 stool from humans. The samples were cultured on cetrimide agar and the plates were incubated aerobically at 37°C for 24 h. The isolates were examined for the presence of the blaKPC , blaOXA-48 , and blaNDM carbapenemase-encoding genes using PCR and investigated for the exotoxin A (toxA) gene. The toxA gene from carbapenem- group resistant isolates was phylogenetically analysed. RESULTS: P. aeruginosa was isolated from buffaloes, cattle, drinking water, and humans, with occurrences of 40%, 34%, 10%, and 20%, respectively. Carbapenem resistance genes were found in 60%, 59%, 67%, and 70% in buffalo, cattle, water and human samples, respectively. The toxA gene was detected in 80% of samples. The phylogenetic analysis showed that cattle and water sequences were in one cluster and more related to each other than to human isolates. CONCLUSION: Occurrence of CRPA among farm animals, drinking water, and humans was high, reflecting the environmental origin of P. aeruginosa and highlighting contaminated water as a potential transmitter of CRPA to livestock and next to humans.

The burden of Coxiella burnetii among aborted dairy animals in Egypt and its public health implications.

Q fever is a zoonotic disease of mounting public health implications. Dairy animals are major reservoir for such disease whereas abortion is the main clinical outcome. The current study was conducted to investigate the burden of C. burnetii abortions among dairy animals in Egypt to provide more knowledge for better control of such disease. For this purpose, placental cotyledons and vaginal discharges from 108 aborted dairy animals (27 sheep, 29 goats, 26 cattle, 26 buffaloes) were examined for the presence of C. burnetii by nested PCR. Serum samples from 58 human contacts were examined for the presence of C. burnetii IgG antibodies using ELISA. Out of the 108 examined animals only one goat yielded positive result in both placental tissue and vaginal discharges with an overall prevalence 0.9% while that among goats is 3.4%. Moreover, the seroprevalence of C. burnetii IgG antibodies among the examined individuals was 19% whereas the prevalence in farmers is significantly higher than that among veterinarians and veterinary assistants. In conclusion, C. burnetii may play a role in dairy goat abortions rather than other dairy animals in Egypt while its public health implications cannot be ruled out.

Norway rat (Rattus norvegicus) as a potential reservoir for Echinococcus granulosus: A public health implication.

Cystic hydatidosis is a re-emerging parasitic zoonosis with worldwide distribution. The current study was carried out to investigate the possible role of rats in the epidemiology of such disease in urban and suburban areas. For this purpose, a total of 50 feral Norway rats (Rattus norvegicus) were collected from urban and suburban settings, Cairo, Egypt. Rats were examined to be infected with cystic hydatidosis through serological examination by IHA test as well as post-mortem examination of internal organs, histopathological or molecular identification of the collected cysts. Moreover, 42 persons inhabiting suburban areas were tested for cystic hydatidosis by IHA. The overall seroprevalence rates of cystic hydatidosis in the examined rats and persons were 36% and 11.9% respectively. Cysts from 3 rats were identified as E. granulosus hydatid cysts (one via histopathological examination while the others by molecular technique and genotyped as G6 strain). The results of the current study highlight the possible role of Norway rat in the epidemiological cycle of E. granulosus especially in urban and suburban settings.

Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt.

This study investigated the occurrence of carbapenem-resistant Klebsiella pneumoniae strains in broiler chickens, drinking water and humans working in contact with chickens and identified the carbapenem resistance determinants among isolates from different sources. Internal organs and droppings were collected from 100 broilers with signs of respiratory disease at five broiler farms in Egypt. Additionally, 20 drinking water samples and 49 faecal samples from workers and veterinarians working at these farms were included. Following culture on MacConkey agar, suspected K. pneumoniae colonies were identified by phenotypic testing. Susceptibility to carbapenems was tested in confirmed K. pneumoniae isolates by disk diffusion. Carbapenem-resistant isolates were subjected to PCR for detection of carbapenemase-encoding genes (blaKPC, blaOXA-48 and blaNDM). K. pneumoniae was isolated from 35% of broilers and 25% of water samples. Of the 35 poultry isolates, 15 were carbapenem-resistant; all of them were blaNDM-positive, including 11 isolates harbouring blaKPC, blaOXA-48 and blaNDM and 4 containing either blaKPC and blaNDM (n=3) or blaOXA-48 and blaNDM (n=1). Similarly, three of five K. pneumoniae isolates from drinking water were positive for blaKPC and blaNDM (n=1) or for all three genes (n=2). Interestingly, 56% of K. pneumoniae from humans displayed carbapenem resistance; all of them were positive for the three carbapenemase genes. Carbapenemase-producing K. pneumoniae occurred at relatively high frequency among broilers, drinking water and workers at poultry farms in Egypt. Additional work is needed to confirm transmission between poultry and humans and to elucidate the direction and mechanism of transmission.

Occurrence of human pathogenic Clostridium botulinum among healthy dairy animals: an emerging public health hazard.

The current study was conducted to investigate the occurrence of human pathogenic Clostridium botulinum in the feces of dairy animals. Fecal samples were collected from 203 apparently healthy dairy animals (50 cattle, 50 buffaloes, 52 sheep, 51 goats). Samples were cultured to recover C. botulinum while human pathogenic C. botulinum strains were identified after screening of all C. botulinum isolates for the presence of genes that encode toxins type A, B, E, F. The overall prevalence of C. botulinum was 18.7% whereas human pathogenic C. botulinum strains (only type A) were isolated from six animals at the rates of 2, 2, 5.8, and 2% for cattle, buffaloes, sheep, and goats, respectively. High fecal carriage rates of C. botulinum among apparently healthy dairy animals especially type A alarm both veterinary and public health communities for a potential role which may be played by dairy animals in the epidemiology of such pathogen.