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.

Molecular detection of Coxiella burnetii in blood and hard tick-infested Egyptian camels and the possibility of coinfections.

Coxiella burnetii, a bacterium that causes Q fever. It can infect mammals and has a global geographical distribution, but data on its occurrence in Egyptian dromedaries and the associated ticks are limited. Therefore, this study aims to detect C. burnetii in the blood of infested camels and associated ticks collected from Egypt by using molecular techniques and to examine the possibility of coinfections with C. burnetii. A total of 133 blood samples and 1260 hard ticks infesting these camels were collected from Egyptian slaughterhouses. Nested PCR and sequencing were used based on the IS1111 gene for molecular detection of C. burnetii. The identification of tick species at the molecular level was performed using the COX1 gene. C. burnetii was detected in Hyalomma (H) dromedarii, H. anatolicum, H. marginatum, Amblyomma (Am) lipidium, and Am. cohaerens with an overall prevalence rate of 1.3% (16/1260), while in the camel blood samples, it was 15.8% (21/133). Out of C. burnetii-positive ticks, there were double infections by Borrelia species and C. burnetii in H. dromedarii and Am. lipidium and triple infections at one Am. cohaerens tick (C. burnetii, Borrelia spp., and Babesia microti). In addition, two positive camel blood samples were found to carry C. burnetii with Borrelia spp. Our research findings indicate the presence of Coxiella burnetii among camels and their associated ticks in Egypt and emphasize the potential of having coinfection. To prevent the transmission of this infection to other animal species or humans, appropriate control measures should be implemented.

Molecular detection of Babesia microti in dromedary camels in Egypt.

Babesia microti (Apicomplexa: Piroplasmida) causes a medically important tick-borne zoonotic protozoan disease. Egyptian camels are susceptible to Babesia infection; however, just a few cases have been documented. This study aimed to identify Babesia species, specifically Babesia microti, and their genetic diversity in dromedary camels in Egypt and associated hard ticks. Blood and hard tick samples were taken from 133 infested dromedary camels slaughtered in Cairo and Giza abattoirs. The study was conducted from February to November 2021. The 18S rRNA gene was amplified by polymerase chain reaction (PCR) to identify Babesia species. Nested PCR targeting the ß-tubulin gene was used to identify B. microti. The PCR results were confirmed by DNA sequencing. Phylogenetic analysis based on the ß-tubulin gene was used to detect and genotype B. microti. Three tick genera were identified in infested camels (Hyalomma, Rhipicephalus, and Amblyomma). Babesia species were detected in 3 out of 133 blood samples (2.3%), while Babesia spp. were not detected in hard ticks by using the 18S rRNA gene. B. microti was identified in 9 out of 133 blood samples (6.8%) and isolated from Rhipicephalus annulatus and Amblyomma cohaerens by the ß-tubulin gene. The phylogenetic analysis of the ß-tubulin gene revealed that USA-type B. microti was prevalent in Egyptian camels. The results of this study suggested that the Egyptian camels may be infected with Babesia spp. and the zoonotic B. microti strains, which pose a potential risk to public health.

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.

Modified dot-ELISA for diagnosis of human trichinellosis.

This study aimed to modify Dot-Enzyme-linked immunosorbent assay (dot-ELISA) for the diagnosis of human trichinellosis and to compare its performance with indirect ELISA and Western-blot assay (EITB). A total of 175 human serum samples were enrolled in the study. Indirect ELISA was used for the primary diagnosis. EITB versus fractionated 1st larval stage excretory-secretory antigens (TL-1 ESA) revealed three specific protein fractions at MW of 45, 50, and 55 kDa (kDa). Dot-ELISA was performed in two ways. In the first one, sera were dotted on the separated three specific protein fractions, while in the second one the three fractions were eluted, concentrated at one pooled antigen that used in classic dot-ELISA. Both types of dot-ELISA proved absolute (100%) sensitivity and specificity in comparison with the gold standard EITB reaction. While sensitivity of ELISA was 100% and its specificity was 79.5%. The fraction at 45 kDa was the most sensitive one. The use of the pooled antigen improved the test results. The described dot-ELISA is an easy applicable diagnostic tool gathering the benefits of both ELISA and EITB.

The Surveillance of Borrelia Species in Camelus dromedarius and Associated Ticks: The First Detection of Borrelia miyamotoi in Egypt.

Tick-borne diseases (TBDs) are emerging and re-emerging infections that have a worldwide impact on human and animal health. Lyme borreliosis (LB) is a severe zoonotic disease caused by the spirochete Borrelia burgdorferi sensu lato (s.l.) transmitted to humans by the bite of infected Ixodes ticks. Borrelia miyamotoi is a spirochete that causes relapsing fever (RF) and is genetically related to Borrelia burgdorferi s.l. However, there have been no reports of B. miyamotoi in Egypt, and the data on LB in camels is scarce. Thus, the present study was conducted to screen and genetically identify Borrelia spp. and B. miyamotoi in Egyptian camels and associated ticks using polymerase chain reaction (PCR). METHODS: A total of 133 blood samples and 1596 adult hard ticks were collected from Camelus dromedaries at Cairo and Giza slaughterhouses in Egypt. Tick species were identified by examining their morphology and sequencing the cytochrome C oxidase subunit 1 (cox1) gene. Borrelia spp. was detected using nested PCR on the IGS (16S-23S) gene, and positive samples were genotyped using 16S rRNA and glpQ spp. genes specific for Borrelia burgdorferi and Borrelia miyamotoi, respectively. The positive PCR products were sequenced and analyzed by phylogenetic tree. RESULTS: Analysis of the cox1 gene sequence revealed that the adult ticks belonged to three genera; Hyalomma (H), Amblyomma (Am), and Rhipicephalus (R), as well as 12 species, including H. dromedarii, H. marginatum, H. excavatum, H. anatolicum, R. annulatus, R. pulchellus, Am. testudinarium, Am. hebraeum, Am. lipidium, Am. variegatum, Am. cohaerens and Am. gemma. Borrelia spp. was found in 8.3% (11/133) of the camel blood samples and 1.3% (21/1596) of the ticks, respectively. Sequencing of the IGS (16S-23S) gene found that B. afzelii, detected from H. dromedarii and H. marginatum, and B. crocidurae, which belongs to the RF group, was detected from one blood sample. B. burgdorferi and B. miyamotoi were discovered in the blood samples and tick species. Phylogenetic analysis of the glpQ gene showed that the B. miyamotoi in this study was of the Asian and European types. CONCLUSIONS: These results suggest that the camels can be infected by Lyme borrelia and other Borrelia bacteria species. This study also provides the first insight into the presence of Borrelia miyamotoi and B. afzelii DNA in camels and associated ticks in Egypt.

Retail chicken giblets contaminated with extended-spectrum cephalosporin- and carbapenem-resistant Salmonella enterica carrying blaCMY-2.

Background and Aim: Chickens are considered as the main source of Salmonella, with infection potentially spreading to the public through outlets. The study aimed to investigate poultry shops for Salmonella enterica resistant to extended-spectrum cephalosporins-resistant (ESCR) and carbapenems-resistant (CR). Materials and Methods: Samples were collected from chicken giblets, water tanks, and workers at retail shops. Salmonella was isolated and serotyped; the presence of invA, stn, ompA, and ompF was determined using polymerase chain reaction (PCR). The isolates were tested for ESCR and CR by a disk-diffusion test; a confirmatory extended-spectrum ß-lactamase (ESBL) test was performed by combinational disk-diffusion test with clavulanic acid. The resistant isolates were screened for ESBL (blaTEM, blaSHV, blaCTX-M, and blaOXA-1), AmpC blaCMY-2, and carbapenemase (blaKPC, blaNDM, and blaOXA-48) genes using PCR. Results: S. enterica was isolated from chicken giblets (13/129) and the 13 isolates were ESCR. Based on the confirmatory ESBL test and CR, the 13 isolates were classified into the following resistance phenotypes: ESBL-producing and CR (n=4), ESBL-producing (n=1), non-ESBL-producing and CR (n=6), and non-ESBL-producing (n=2). All the five isolates with ESBL-producing phenotype carried predominantly blaTEM, blaSHV, and blaCMY-2. Regardless of being phenotypically CR, none of these isolates carried any of the tested carbapenemase genes. Surprisingly, the isolates with non-ESBL phenotype were found to carry blaTEM, blaSHV, and blaCMY-2. The blaKPC was present mainly in the isolates with non-ESBL and CR phenotypes. Interestingly, two isolates of the non-ESBL and CR phenotype showed resistance to cefepime, the fourth generation cephalosporins. Salmonella was also recovered from the water tanks (2/7) and the workers (2/16). The four isolates were ESCR and showed a non-ESBL-producing and CR phenotype; they harbored blaTEM, blaSHV, blaOXA-1, and blaKPC. The blaCMY-2 was found in one isolate from water and one from humans. All Salmonella isolates carried invA, stn, ompA, and ompF. Conclusion: Virulent ESCR S. enterica were identified in retail shops. The isolates carried blaCMY-2 and ESBL-genes, with a high proportion showing CR. Transmission of such strains to humans through food leads us to recommend regular inspection of retail outlets for antibiotic-resistant bacteria.

Migratory birds as a potential overseas transmitter of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) has a great public health importance. This study was conducted to investigate the potential role of migratory birds in the transmission of STEC. For this purpose, cloacal swabs were collected from 349 migratory birds (209 ducks and 140 quails) from Damietta governorate, Egypt. The collected swabs were cultured for isolation of STEC using the STEC CHROMagar. STEC isolates were identified based on colonial characteristics, Gram's stain, conventional biochemical tests and molecular detection of stx1, stx2 and eae genes. Positive isolates were serotyped and examined for their antibiotic susceptibility pattern. Furthermore, gene sequencing was performed for genes stx1and stx2. Of the examined birds, two STEC isolates were a obtained with an overall occurrence rate 0.57% (2/349), one isolate carried stx2 gene from a migratory quail 0.71% (1/140), and another isolate from a migratory duck carried stx1 gene 0.48% (1/209), whereas both isolates were negative for eae gene. Moreover, the duck isolate was serotyped O86, while the quail isolate was serotyped O125; both isolates were multidrug resistant. The phylogenetic analysis of the obtained stx1 and stx2 genes revealed high genetic relatedness to those isolated from human cases in the countries where such birds either lived or were in their migratory pathway. In conclusion, this study highlights the potential role of migratory birds in transmitting multidrug-resistant STEC across their migratory pathway.

Extended-spectrum ß-lactamase-producing Salmonella serovars among healthy and diseased chickens and their public health implication.

OBJECTIVES: This study investigated the occurrence of extended-spectrum ß-lactamase (ESBL)-producing Salmonella and the associated virulence genes among farmed chickens. METHODS: Cloacal swab samples were collected from apparently healthy and diseased chickens and were cultured for Salmonella using conventional methods. The isolates were serotyped using slide agglutination tests and were examined by polymerase chain reaction (PCR) for the virulence genes invA, stn, svpC and pefA and the outer membrane protein-encoding genes ompA and ompF. Screening for ESBL resistance was performed using the disk-diffusion test, the combinational-disk test with clavulanic acid, and multiplex PCR for blaTEM, blaSHV, blaCTX-M and blaOXA. The presence of the AmpC blaCMy-2 was tested among the ESBL-negative isolates by uniplex PCR. The resistant isolates were partially sequenced based on the stn gene. RESULTS: The Salmonella isolation rate was 3.4% (6/175) from healthy and 11.1% (14/126) from diseased chickens. The 20 isolates belong to serotypes with public health significance like Typhimurium, Kentucky and Infantis. All the isolates possess invA, stn, svpC and ompF genes; 16 isolates harboured ompA, and one carried pefA. Of the 20 isolates, 19 were resistant to more than one antibiotic. Of these 19 isolates, 16 were ESBL-producing with the majority carrying blaTEM and blaSHV genes. The four ESBL-negative isolates carried blaCMY-2. Partial-stn-sequencing of the isolates revealed a high genetic relatedness to Salmonella strains from patients in Egypt and Asia. CONCLUSIONS: Virulent ESBL-producing Salmonella was isolated from healthy and diseased chickens; the strains have a close relationship to human strains, posing a public health threat.

Evidence of Zoonotic Transmission of Helicobacter canis Between Sheep and Human Contacts.

Helicobacter species are newly emerging bacteria with great public implications but till now its epidemiology is not fully understood; so, this study was conducted to investigate the possible role of ruminants in the epidemiology of these pathogens. For this purpose, fecal samples were collected from 149 animals (76 sheep, 33 goats, 21 cattle, and 19 buffaloes) and stool specimens from 10 animal caretakers in intimate contact with the examined animals. All samples were examined for the presence of Helicobacter species through detection of Helicobacter genus specific 16S rRNA using PCR. Then, all positive Helicobacter spp. amplicons were sequenced to recognize their species through BLAST analysis at GenBank. The overall prevalence of Helicobacter spp. was 14.8% while the distribution among the different animals was 26.3%, 3%, 4.8%, and 0% in sheep, goats, cattle, and buffaloes respectively. Helicobacter canis was the predominant species and detected only in sheep (21%) and goats (3%). Moreover, Helicobacter winghamensis and Helicobacter canadensis were also detected in sheep but not in other animals, whereas the only positive bovine sample was identified as Helicobacter bovis. On the other hand, 4 out of 10 humans were positive for Helicobacter spp. and all sequences were identified as H. canis. The sequences identity matrix and phylogenetic analysis of H. canis sequences from humans and sheep contacts revealed that one human sequence was identical to that of sheep and making sister group clade, which prove the zoonotic transmission of this pathogen between sheep and human contacts. However, our findings highlight sheep as a potential reservoir for H. canis, further researches are needed to address the potential role of sheep in the food-borne transmission of such emerging pathogen.

Saliva as an easy specimen for diagnosis of human and animal fascioliosis.

In the present study saliva was evaluated for detection of human and animal fascioliosis. Both saliva and serum samples were collected from 12 Fasciola infected patients, 17 cattles and 20 sheep harboring Fasciola eggs only in their faeces. Samples from negative non-infected hosts were also collected. Experimental infection by F. gigantica in rabbits was carried out for determination of the first time appearance of anti-F. gigantica antibodies (AFAb) and circulating F. gigantica antigen (CFAg). This was carried out by indirect and sandwich ELISA using purified antigen (26-28 KD) and monoclonal antibodies. AFAb were detected in saliva of naturally infected patients, cattles and sheep, the sensitivity of the assays reached 66.6%, 64.7% and 65% respectively, while the sensitivity using serum samples was 91.66, 94.11 & 100% respectively. In the contrary AFAb in saliva was more specific (100%) than that in serum as it was 100%, 92.0% and 96.0% in humans, cattle and sheep respectively. CFAg showed higher sensitivity in diagnosis using saliva in comparison with AFAb as it was 83.3, 76.47 & 85% in patients, cattles & sheep respectively. Similarly, the specificity of CFAg in saliva was higher than that recorded using serum samples as it was 100%, 96.0% and 96.0% in the three groups respectively. AFAb and CFAg were detected in serum of experimentally F. gigntica infected rabbits at the end of the first week post infection, and in saliva at the 15th and 18th day post infection. These data introduce saliva as an easily collected sample that can be used for diagnosis of zoonotic fascioliosis.

Evaluation of Capillaria philippinensis coproantigen in the diagnosis of infection.

In order to overcome the false negative diagnosis of infection with C. philippinesis at time of absence of eggs in stool, coproantigen prepared from stools of infected patients was evaluated serologically. This antigen was able to detect anti-Capillaria antibodies in the sera of infected cases at the same OD level produced with Capillaria crude worm antigen using indirect ELISA technique C. philippenensis coproantigen did not cross-react with sera from patients with schistosomiasis mansoni, fascioliasis or strongyloidiasis at 1:00 serum dilution. Laboratory-prepared hyperimmune sera versus crude worm antigen of C. philippinensis succeeded in capturing Capillaria antigen prepared from the stools of infected patients and did not cross react with coproantigens prepared from stool samples of cases infected with S. mansoni or Fasciola using sandwich ELISA technique.