Detection of TEM and CTX-M Genes in Escherichia coli Isolated from Clinical Specimens at Tertiary Care Heart Hospital, Kathmandu, Nepal

 Multidisciplinary Digital Publishing Institute (MDPI)

 

Diseases. 2021 Feb 7;9(1):15. DOI: 10.3390/diseases9010015. PMID: 33562276

 

Detection of TEM and CTX-M Genes in Escherichia coli Isolated from Clinical Specimens at Tertiary Care Heart Hospital, Kathmandu, Nepal

Ram Shankar Prasad Sah ¹, Binod Dhungel ¹, Binod Kumar Yadav ², Nabaraj Adhikari ¹, Upendra Thapa Shrestha ¹, Binod Lekhak ¹, Megha Raj Banjara ¹, Bipin Adhikari ³, Prakash Ghimire ¹, Komal Raj Rijal ^1*

 

¹ Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu 44618, Nepal

² Shahid Gangalal National Heart Centre, Bansbari, Kathmandu 44618, Nepal

³ Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand

 

* Author to whom correspondence should be addressed.

 

ABSTRACT

Background: Antimicrobial resistance (AMR) among Gram-negative pathogens, predominantly ESBL-producing clinical isolates, are increasing worldwide. The main aim of this study was to determine the prevalence of ESBL-producing clinical isolates, their antibiogram, and the frequency of ESBL genes (bla_TEM and bla_CTX-M) in the clinical samples from patients.

Methods: A total of 1065 clinical specimens from patients suspected of heart infections were collected between February and August 2019. Bacterial isolates were identified on colony morphology and biochemical properties. Thus, obtained clinical isolates were screened for antimicrobial susceptibility testing (AST) using modified Kirby-Bauer disk diffusion method, while ESBL producers were identified by using a combination disk diffusion method. ESBL positive isolates were further assessed using conventional polymerase chain reaction (PCR) to detect the ESBL genes bla_TEM and bla_CTX-M.

Results: Out of 1065 clinical specimens, 17.8% (190/1065) showed bacterial growth. Among 190 bacterial isolates, 57.4% (109/190) were Gram-negative bacteria. Among 109 Gram-negative bacteria, 40.3% (44/109) were E. coli, and 30.2% (33/109) were K. pneumoniae. In AST, 57.7% (n = 63) Gram-negative bacterial isolates were resistant to ampicillin and 47.7% (n = 52) were resistant to nalidixic acid. Over half of the isolates (51.3%; 56/109) were multidrug resistant (MDR). Of 44 E. coli, 27.3% (12/44) were ESBL producers. Among ESBL producer E. coli isolates, 58.4% (7/12) tested positive for the bla_CTX-M gene and 41.6% (5/12) tested positive for the bla_TEM gene.

Conclusion: Half of the Gram-negative bacteria in our study were MDR. Routine identification of an infectious agent followed by AST is critical to optimize the treatment and prevent antimicrobial resistance.

Keywords: Cefotaximase; ESBL; Nepal; Temoneira (TEM); antimicrobial resistance; blaCTX-M; blaTEM; uropathogenic E. coli.

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Amoebiasis

 Amoebiasis is an infection of human gastrointestinal tracts by a protozoan, Entamoeba histolytica which starts invading the intestine and then to the other organs including liver, lungs, brain, and spleen etc. According to WHO, it is one of the fourth leading causes of mortality accounting for 70 thousand deaths among protozoal infections after malaria, Chagas disease, and leishmaniasis. It also causes significant morbidity to the patients after malaria and trichomoniasis. The degree of pathogenesis and diseases outcome by Entamoeba histolytica depends mainly on three major factors such as parasite, host and environmental.

 

Cysts and trophozoites are passed in feces 1 . Cysts are typically found in formed stool, whereas trophozoites are typically found in diarrheal stool. Infection with Entamoeba histolytica (and E. dispar) occurs via ingestion of mature cysts 2 from fecally contaminated food, water, or hands. Exposure to infectious cysts and trophozoites in fecal matter during sexual contact may also occur. Excystation 3 occurs in the small intestine and trophozoites 4 are released, which migrate to the large intestine. Trophozoites may remain confined to the intestinal lumen (A: noninvasive infection) with individuals continuing to pass cysts in their stool (asymptomatic carriers). Trophozoites can invade the intestinal mucosa (B: intestinal disease), or blood vessels, reaching extraintestinal sites such as the liver, brain, and lungs (C: extraintestinal disease). Trophozoites multiply by binary fission and produce cysts 5, and both stages are passed in the feces 1. Cysts can survive days to weeks in the external environment and remain infectious in the environment due to the protection conferred by their walls. Trophozoites passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric environment (https://www.cdc.gov/dpdx/amebiasis/index.html)

Pathogenesis:

Pathogen factors: E. histolytica has many virulence factors including lectin, amoebic pore forming proteins, many different kinds of proteases, antiphagocytic mechanism of trophozoites etc.  After adherence to epithelial mucous layer through Gal/GalNAc lectin, it produces glycosidases and proteases which degrade the epithelial lining of human small intestine.  E. histolytica then produces many different kinds of cysteine proteases to overcome pathogenesis and host immune response. EhCP4 degrades sIgA, EhCP5 cuts to form amoeba pore, EhCPLf disrupts the tight junctions and so on. Likewise, CXXC rich proteins have erythrophagocytosis activities. These cysteine proteases play 70-80% role in pathogenesis. The trophozoites then interact with intestinal epithelial linings and other host tissues inducing apoptosis. The more is the expression of such virulence factors; the more is the severity of disease.  Hence the burden also depends on the expression level of different virulent genes on the host body by different strains. Some strains also gain resistant to antiprotozoal drugs which can be more pathogenic. The emerging new strains of the protozoa is more virulent and the infection caused by such strains are difficult to manage.

Host factors: The first defence lining of host is anti-lectin IgA circulating in their gut which can easily avoid the symptomatic infections by E. histolytica. These sIgA neutralizes the protozoa preventing excystation. The adherence of Gal/GalNAc lectin induces the production of IL-2 and gamma interferon in T-cell and inducible nitric oxide synthase through TNF alpha production. These immune responses provide the protection against the colitis and abscess in amoebic infections. The host who are pre-exposed may have already preformed immune responses and avoid the symptomatic infections. Besides, one of the studies also reported the higher prevalence of amoebic liver abscess among males than in females due to lack of sufficient gamma interferon in their serum. Likewise, the immunocompetent population have less chance of symptomatic infections whereas in immunocompromised individuals like HIV/AIDS, malnourished children, organ transplant patients and those having chronic infections with heart, pulmonary and liver may have severe amoebic infections with higher rate of mortality and morbidity.

Environment in gut: The role of microbiome in gut of human is another important factor in E. histolytica pathogenesis and diseases outcomes. E. histolytica normally grazes on intestinal bacteria for nutrients especially gram-negative enteric bacteria including Escherichia coli and Shigella dysenteriae. One of the studies from Northern India reported that the decreased microbial load in Bacteroides, Clostridium coccidias sub-group, Clostridium leptum sub-group Lactobacillus, Campylobacter and Eubacterium among amoebic patients while Bifidobacterium load is increased. The ingestion of few of these bacteria enhance the over expression of lectin, a major virulent factor in E. histolytica which increases cytopathic effect. Children with Prevotella copri as microbiome in the gut have more common E. histolytica related diarrhea while those with Clostridia related bacteria are more resistant to E. histolytica infection.

Others: The severity and number of cases also depends on geographical distribution, socioeconomic status and personal hygiene. Although it is a world widely distributed, a higher number of diseases occur in the developing and underdeveloped countries due to contaminated drinking water supplies. The lack of unawareness, ignorance, overpopulated, poor sanitation and poor hygiene in food preparation in such low-income countries are major predisposing factors for burden and outcomes of diseases among those communities.  An increased occurrence of E. histolytica infections are observed among those who practice homosexual activities and oro-anal sex. Moreover, they present severe symptoms than those of immunocompetent individuals.  Individuals with habitual eating out-side foods may also have a higher rate of amoebic diarrhoea rather than direct exposure to human and animal excreta. Few vectors including flies play a significant role in the transmission of amoebic cysts to such open food products.

 

Clinical manifestations of amoebic liver abscess

Over 90% of amoebiasis is asymptomatic, while only 4-10% of infected patients present different kinds of clinical signs and symptoms. As the colonization of Entamoeba histolytica starts from the intestine, the majority of cases are related to intestinal amoebiasis showing diarrhea, dysentery, colitis as general syndromes. However, E. histolytica can also disseminate to other organs through the portal blood circulation causing extraintestinal infections. Among the invasive amoebiasis, 10-15% of adults develop amoebic liver abscess, which is one of the most common extraintestinal infections by E. histolytica. The disease is a persistently progressive and may lead to fatality. However, the rate of fatality decreases with advances in treatment and diagnostic procedures. The amoebic liver abscess starts with spreading of hematophagous trophozoites from colonic mucosa via the portal circulation. The individuals with amoebic liver abscess hence also present amoebic colitis but amoebic dysentery like symptoms as in intestinal amoebiasis is absent. Even no trophozoites and cysts form of E. histolytica is observed under stool microscopy. The infected persons have a fever with chills and profuse sweating in the afternoon and night. The patients also have abdominal pain in the upper quadrant, especially in right hypochondrium. The pain may increase when an individual deeply breathes, cough, and step right foot on walking. The individual with epigastric pain feels intense pain in the liver region during digital pressure and fist percussion.

The patients having single and multiple abscesses in the liver suffer hepatic tenderness and painful hepatomegaly. However, around 8% of individual with liver abscess have mild jaundice, unless complication of multiple abscesses occurs.  Other general symptoms are anorexia, nausea, vomiting, fatigue, and weight loss. The symptoms may last for less than 10 days however, in chronicity, anorexia and weight loss are very common.

Besides, a few pulmonary like symptoms such as cough, dullness, and rales in the right lung base may be present. During amoebic liver abscess, leukocytosis without eosinophilia, mild anemia, raised concentration of alkaline phosphatase and high rate of RBC sedimentation are common clinical findings used in the diagnosis of it.

“Natural Vector of Leishmania in Thailand”

Leishmaniasis, a tropical neglected disease, is a vector-borne disease caused by protozoan parasites of genus Leishmania. The parasites are carried only by female sandflies as the female flies need blood for the development of eggs.  They become infected with the Leishmania parasites while they suck blood from an infected person or animal and then transmit it to another host. Of 900 species of the sand fly, over 70 species are found to be associated with the transmission of leishmaniasis. One of the major vectors for leishmaniasis is the Phlebotomine sandfly. There are around 500 known Phlebotomine species, however, nearly 30 species are capable to transmit leishmaniasis ( https://www.who.int/leishmaniasis/en/).

 

In order to be incriminating natural vectors, they should have the following criteria. The wild females not having recent blood meal (<36 hrs) should contain promastigotes of Leishmania parasites. The anterior midgut of infected females sand flies must have infective forms of Leishmania. The flies should be attracted to and bite humans and other reservoir hosts. They have to be strongly associated with humans and any reservoir host and finally, the experimental transmission is achieved after infection from natural host species or equivalent laboratory model. To date, no published data have been documented to verify the vectors found in Thailand to prove as incriminating natural vectors according to the criteria mentioned above. Few studies have reported the presence of Leishmania DNA in female sandflies collected from different provinces of Thailand by using molecular tools. Similarly, few investigators have studied the natural habitats of the sandflies and their related host. On the basis of these characteristics, they have suspected these vectors as the potential natural vectors of leishmaniasis.

 

Chamnarn and team had collected 2401 Phlebotomine sand flies from 16 limestone caves (temperature range 26-28°C) in Kanchanaburi province, Thailand, and identified them following standard protocol. The study had updated to a total number of 26 species of sandflies belonged to the four genera Sergentomyia, Phlebotomus, Nemopalpus, and Chinius in Thailand. These are C. barbazani, N. vietnamensis, P. asperulus, P. barguesae, P. betisi, P. hoepplii, P. major major, P. mascomai, P. philippinensis gouldi, P. pholetor, P. stantoni, P. teshi, S. anodontis, S. bailyi, S. barraudi, S. brevicaulis, S. dentata, S. gemmea, S. hodgsoni hodgsoni, S. indica, S. iyengari, S. perturbans, S. phasukae, S. punjabensis, S. quatei and S. sylvatica. The most frequent cave species found in this study were P. major major and S. anodontis. The human biting species, P. major was also the first time reported from this study. They have observed ecological habitats and behaviors (host feeding, biting activity, resting areas at day time, sheltering places at night) of sandflies to identify. However, they are not concerned about whether there was a presence of Leishmania infective forms in their midguts or not. They only proposed the sand flies as potential natural vectors for leishmaniasis (Apiwathnasorn et al., 2011). Late one more species, S. mahadevani had been identified, and altogether 27 species have been reported date in Thailand to date.

 

Among these potential vectors, different studies have confirmed different species that transmit the Leishmania parasites. Kanjanopas and team collected sandflies from individual households in Hat Samran District, Trang Province, southern Thailand where coinfection of visceral leishmaniasis and Human Immunodeficiency Syndrome (HIV) had been reported. They identified the female sandflies with the help of Entomologists and finally sent them in Molecular laboratory in Taiwan. They have evaluated for natural infections with L. siamensis confirmed by amplifying heat shock protein 70 (hsp70) of Leishmania parasite by PCR method. Although other criteria had not been studied, S. (Neophlebotomus) gemmea is considered as a potential natural vector for L. siamensis (Kanjanopas et al., 2013).

 

Chusri et al. carried out active human case surveys processing blood, saliva and urine samples from 99 villagers living in an affected area in Na Thawi District. The team had also studied details of animal reservoirs including blood samples from dogs, cats, black rats, and Indochinese ground squirrels. Sandflies were collected from villagers’ houses and plantation which were identified at office of Disease Prevention and Control. The presence of Leishmania parasite in those sandflies were confirmed by amplifying parasite specific 18s rRNA followed by nucleotide sequencing. The study finally reported female S. (Neophlebotomus) gem­mea and female S. (Parrotomyia) barraudi were potential natural vectors for L. siamensis (Chusri et al., 2014).

 

Another study by Sukra and the team had reported six sand fly vectors of Sergentomyia; S. gemmea, S. iyengari, S. barraudi, S. indica, S. silvatica and S. perturbans as potential natural vectors of leishmaniasis. The team had collected sandflies from three provinces of Thailand; Phang-nga, Suratthani, and Nakonsitammarat. They had trapped the flies at 200m around the patients’ houses by CDC light traps. The traps had also been placed in other possible habitats such as cattle corrals, pig sites, stacks of leaves etc. These flies were then identified, however, their role in the transmission of Leishmania parasites was not confirmed. One of the important vectors of leishmaniasis of genus Phlebotomus, Phlebotomus argentipes, was also detected. They suspected them as potential vectors because they were found in the infected areas (Sukra et al. 2013).

 

Leishmaniasis cases in Thailand constituted only imported cases before 1999. The recent studies emphasizing indigenous leishmaniasis identify two new species, L. siamnesis and L. martiniquensis as autochthonous species among Thai patients. As reported by Chusri et al., 2014 and Kanjanopas et al., 2013, S. (Neophlebotomus) gemmea and S. (Parrotomyia) barraudi as could serve as potential vectors for L. martiniquensis (Leelayoova et al., 2017).

 

Srisuton et al. collected sand flies from endemic areas (Songkhla and Phatthalung Provinces) and non-endemic area (Chumphon Province) of leishmaniasis in Thailand. Head and genitalia dissection of pre-identified female sandflies were done for morphology identification, and the remaining parts were used to detect Leishmania and Trypanosoma DNA. One new vector identified as S. khawi was found to carry Leishmania and Trypanosoma parasites. The vector species was confirmed as a potential natural vector capable of transmitting L. martiniquensis in the human population (Srisuton et al., 2019).

 

References:

Apiwathnasorn C, Samung Y, Prummongkol S, Phayakaphon A, and Panasopolkul C. Cavernicolous species of phlebotomine sand flies from Kanchanaburi province, with an updated species list for Thailand. Southeast Asian J Trop Med Public Health. 2011; 42 (6): 1405-1409.

Chusri S, Thammapalo S, Silpapojakul K, Siriyasatien P. Animal reservoirs and potential vectors of Leishmania siamensis in southern Thailand. Southeast Asian J Trop Med Public Health. 2014; 45 (1): 13-19.

Kanjanopas K, Siripattanapipong S, Ninsaeng U, Hitakarun A, Jitkaew S, Kaewtaphaya P, et al. Sergentomyia (Neophlebotomus) gemmea, a potential vector of Leishmania siamensis in southern Thailand. BMC Infectious Diseases.; 2013 13: 333.

Leelayoova S, Siripattanapipong S, Manomat J, Piyaraj P, Tan-ariya P, Bualert L, et al. Leishmaniasis in Thailand: A Review of Causative Agents and Situations. Am J Trop Med Hyg. 2017; 96 (3): 534–542. doi:10.4269/ajtmh.16-0604.

Srisuton P, Phumee A, Sunantaraporn S, Boonserm R, Sor-suwan  S, Brownell  N, et al. Detection of Leishmania and Trypanosoma DNA in Field-Caught Sand Flies from Endemic and Non-Endemic Areas of Leishmaniasis in Southern Thailand. Insects. 2019; 10: 238; doi:10.3390/insects10080238.

Sukra K, Kanjanopas K, Amsakul S, Rittaton V, Mungthin M, Leelayoova S. A survey of sandflies in the affected areas of leishmaniasis, southern Thailand. Parasitol Res. 2013; 112: 297–302. DOI 10.1007/s00436-012-3137-x.

https://www.who.int/leishmaniasis/en/