Highly Colistin-resistant Aeromonas jandaei from a Human Blood Sample.

Aeromonas species are Gram-negative rods known to cause infections such as gastroenteritis, bacteremia and wound infections. Colistin is one of few treatments for multidrug-resistant Gram-negative bacteria. However, colistin-resistant bacteria carrying the mobilized colistin resistance (mcr) gene are a threat in healthcare settings worldwide. In recent years, colistin-resistant Aeromonas species have been detected in environmental and clinical samples. We analyzed the genomic characteristics of one highly colistin-resistant A. jandaei isolated from a blood sample in Nepal, which harbored four novel mcr-like genes on its chromosome. Our study strongly suggests that A. jandaei is a reservoir of colistin-resistant genes. Inappropriate use of drugs in medicine and food production should be reduced and continued global surveillance for colistin-resistant bacteria is necessary.

Stenotrophomonas maltophilia from Nepal Producing Two Novel Antibiotic Inactivating Enzymes, a Class A ß-Lactamase KBL-1 and an Aminoglycoside 6'-N-Acetyltransferase AAC(6')-Iap.

Seven drug-resistant strains of Stenotrophomonas maltophilia were isolated from patients at two university hospitals in Nepal. S. maltophilia JUNP497 was found to encode a novel class A ß-lactamase, KBL-1 (Kathmandu ß-lactamase), consisting of 286 amino acids with 52.98% identity to PSV-1. Escherichia coli transformants expressing blaKBL-1 were less susceptible to penicillins. The recombinant KBL-1 protein efficiently hydrolyzed penicillins. The genomic environment surrounding blaKBL-1 was a unique structure, with the upstream region derived from strains in China and the downstream region from strains in India. S. maltophilia JUNP350 was found to encode a novel 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iap, consisting of 155 amino acids with 85.0% identity to AAC(6')-Iz. E. coli transformants expressing aac(6')-Iap were less susceptible to arbekacin, amikacin, dibekacin, isepamicin, neomycin, netilmicin, sisomicin and tobramycin. The recombinant AAC(6')-Iap protein acetylated all aminoglycosides tested, except for apramycin and paromomycin. The genomic environment surrounding aac(6')-Iap was 90.99% identical to that of S. maltophilia JV3 obtained from a rhizosphere in Brazil. Phylogenetic analysis based on whole genome sequences showed that most S. maltophilia isolates in Nepal were similar to those isolates in European countries, including Germany and Spain. IMPORTANCE The emergence of drug-resistant S. maltophilia has become a serious problem in medical settings worldwide. The present study demonstrated that drug-resistant S. maltophilia strains in Nepal harbored novel genes encoding a class A ß-lactamase, KBL-1, or a 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iap. Genetic backgrounds of most S. maltophilia strains in Nepal were similar to those in European countries. Surveillance of drug-resistant S. maltophilia in medical settings in Nepal is necessary.

Emergence of a highly colistin-resistant Aeromonas jandaei clinical isolate harbouring four genes encoding phosphoethanolamine transferases in Nepal.

OBJECTIVES: This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases. METHODS: An isolate of colistin-resistant A. jandaei was obtained from a blood sample of an inpatient in a hospital in Nepal, and its complete genome sequence was determined. Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) transformants expressing genes encoding novel phosphoethanolamine transferase variants were constructed and colistin-susceptibility profiles were determined. RESULTS: The isolate harboured four genes encoding phosphoethanolamine transferases on the chromosome, which were designated eptAv3.2, eptAv3.3, eptAv3.4 and eptAv7.2. The amino acid sequences of EptAv3.2, 3.3 and 3.4 were > 80% identical to MCR-3.1, and that of EptAv7.2 was > 79% identical to MCR-7.1. E. coli expressing eptAv3.2, 3.3 and 3.4 showed reduced susceptibility to colistin, whereas E. coli expressing eptAv7.2 did not. In contrast, A. hydrophila expressing eptAv7.2 showed reduced susceptibility to colistin, whereas A. hydrophila expressing eptAv3.2, 3.3 and 3.4 did not; eptAv3.3 and 3.4 formed a tandem structure. The genomic environments surrounding eptAv3.2, 3.3 and 3.4 were similar to Aeromonas veronii obtained from the effluent of a treatment plant in Japan in 2018. The genomic environment surrounding eptAv7.2 was similar to that of A. jandaei obtained from a chicken in the USA in 2019. CONCLUSIONS: The highly colistin-resistant A. jandaei clinical isolate harboured four chromosomal genes encoding phosphoethanolamine transferases, suggesting that Aeromonas spp. harbouring eptAv genes with strong similarities to mcr-3 and mcr-7 are emerging in medical settings as well as environments.

Molecular characterisation of carbapenem-resistant Pseudomonas aeruginosa clinical isolates in Nepal.

OBJECTIVES: The emergence of carbapenem-resistant Pseudomonas aeruginosa has become a serious worldwide medical problem. The aim of this study was to determine the genetic and epidemiological properties of carbapenem-resistant P. aeruginosa strains isolated from hospitals in Nepal. METHODS: A total of 43 carbapenem-resistant P. aeruginosa isolates obtained from patients in two hospitals in Nepal between 2018 and 2020 were analysed. Their whole genomes were sequenced by next-generation sequencing. A phylogenetic tree was constructed from single nucleotide polymorphism (SNP) concatemers. Multilocus sequence typing (MLST) was performed and antimicrobial resistance genes were identified. RESULTS: Of the 43 isolates, 17 harboured genes encoding carbapenemases, including IMP-1, IMP-26, KPC-2, NDM-1, VIM-2 and VIM-5, and 12 harboured genes encoding 16S rRNA methylases, including RmtB4 and RmtF2. The carbapenem-resistant P. aeruginosa isolated in Nepal belonged to various sequence types (STs), including ST235 (5 isolates), ST244 (7 isolates), ST274 (1 isolate), ST357 (10 isolates), ST654 (3 isolates), ST664 (1 isolate), ST773 (1 isolate), ST823 (3 isolates), ST1047 (8 isolates), ST1203 (2 isolates) and ST3453 (2 isolates). CONCLUSION: To the best of our knowledge, this is the first molecular epidemiological analysis of carbapenem-resistant P. aeruginosa clinical isolates from Nepal. The findings strongly suggest that P. aeruginosa isolates producing carbapenemases and 16S rRNA methylases have spread throughout medical settings in Nepal.

Highly multidrug-resistant Morganella morganii clinical isolates from Nepal co-producing NDM-type metallo-ß-lactamases and the 16S rRNA methylase ArmA.

Morganella morganii can harbour extended-spectrum ß-lactamases and carbapenemases, resulting in increased resistance to multiple antibiotics and a high mortality rate. This study describes the emergence of highly multidrug-resistant clinical isolates of M. morganii from Nepal co-producing NDM-type metallo-ß-lactamases, including NDM-1 and NDM-5, and the 16S rRNA methylase ArmA. This is the first report of M. morganii clinical isolates from Nepal co-producing NDM-1/-5 and ArmA. It is important to establish infection control systems and effective treatments against multidrug-resistant M. morganii.

Emergence of clinical isolates of highly carbapenem-resistant Klebsiella pneumoniae co-harboring blaNDM-5 and blaOXA-181 or -232 in Nepal.

OBJECTIVES: The aim of this study was to describe the emergence in Nepal of clinical isolates of Klebsiella pneumoniae harboring both blaNDM-5 and blaOXA-181/-232. METHODS: Six clinical isolates of K. pneumoniae highly resistant to carbapenems and aminoglycosides were obtained from inpatients in Nepal. Their whole genomes were sequenced by a next generation sequencer. RESULTS: The minimum inhibitory concentrations of meropenem, amikacin and ciprofloxacin were ≥128 µg/ml, >1024 µg/ml and ≥256 µg/ml, respectively. All six isolates co-harbored blaNDM-5, blaOXA-181 or -232 and rmtB. Of them, 1 also harbored rmtF. The blaNDM-5, blaOXA-232 and rmtB in all six isolates were located on plasmids. Of the six isolates tested, one isolate harbored two copies of blaOXA-181 and rmtF on the chromosome. CONCLUSIONS: This is the first report of clinical isolates of K. pneumoniae co-harboring blaNDM-5, blaOXA-181 or -232 and rmtB in Nepal. These strains were highly carbapenem- and aminoglycoside-resistant, and belonged to ST147 or ST395. Of them, ST147 isolate harbored two copies of blaOXA-181 on the chromosome.

Hospital based surveillance and molecular characterization of rotavirus in children less than 5 years of age with acute gastroenteritis in Nepal.

BACKGROUND: Rotavirus remains a significant causative agent of childhood acute gastroenteritis, particularly among children less than 5 years of age. Although precise data on childhood mortality associated with diarrheal disease in Nepal is not available, it is estimated that22% of all rotavirus deaths globally occurs in neighboring country of India. In spite of the substantial burden of rotavirus gastroenteritis in the Indian subcontinent, rotavirus vaccine has not been introduced in Nepal. Continuous surveillance for monitoring rotavirus disease burden and molecular characterization is needed prior to rotavirus vaccine introduction in Nepal. METHODS: A total of 3310 stool samples (2849 hospitalized cases and 461 non-hospitalized cases), were collected from patients <5 years of age from January 2013 to December 2016 and tested for rotavirus antigen by ELISA (ProSpecT, USA). A subset of ELISA positive stool samples was genotyped. Demographic data were collected. RESULTS: During the four-year surveillance period, the overall burden of rotavirus infection was 24% among hospitalized children which was much higher than among non-hospitalized children (12%). The majority of children hospitalized with rotavirus gastroenteritis were less than 2 years of age (86%). Rotavirus-associated gastroenteritis hospitalizations occur year-round in Nepal, but a distinct peak in winter (up to 40% among hospitalized) was observed. Of 735 ELISA positive samples, 492 were genotyped by RT-PCR. The most prevalent genotype was G12P[6] (45.3%), followed byG2P[4](12.2%), G1P[8] (9.6%), G9P[4](7.3%), and G9P[8](4.5%). Mixed infection accounted for 4.4% of cases, 6.2% were partially typed and 10.5% of the samples were G and P untypable. CONCLUSIONS: A high burden of rotavirus gastroenteritis and a diversity of circulating rotavirus strains in Nepal were observed. Recommendation to introduce a rotavirus vaccine with known vaccine effectiveness would help in reducing the severity of Rotavirus diarrheal disease in children less than 5 years of age.

Cyclospora Infection among School Children in Kathmandu, Nepal: Prevalence and Associated Risk Factors.

BACKGROUND: The intestinal coccidian protozoa Cyclospora cayetanensis has emerged as an important cause of parasitic diarrhea among children living in developing countries. This study aimed to determine the prevalence of Cyclospora among the school children of Kathmandu with reference to various associated risk factors. METHODOLOGY: A total of five hundred and seven stool samples from students between the age of 3-14 years, studying in 13 different schools in Kathmandu were collected during the study period (May-November, 2014) and processed at the Public Health Research Laboratory, Institute of Medicine, Kathmandu, Nepal. A modified acid fast staining technique (Kinyoun's method) was used to detect oocyst of Cyclospora from the formal-ether concentrated stool samples. RESULTS: Cyclospora was detected in 3.94% (20/507) of the stool samples examined. The prevalence was found to be highest among the students in the 3-5 year age group i.e. 10.15% (13/128), peaking during the rainy season (June-August). The detection rate was found to be significantly higher (p < 0.05) among children presenting with diarrheal symptoms, household keeping livestock and consumers of raw vegetables/fruits, showing a prevalence of 10.57% (11/104), 10.11% (9/89) and 7.25% (14/193) respectively. CONCLUSION: Consumption of untreated drinking water, fresh produce (raw fruits/vegetables) without proper washing and the presence of livestock at home were found to be predisposing factors for higher susceptibility of infection due to Cyclospora. This finding confirms the existence of a public-health issue with potentially serious consequences whereby children can be infected through exposure to oocysts in contaminated food and water and get ill as a result.

Prevalence of group A genotype human rotavirus among children with diarrhoea in Nepal, 2009-2011.

BACKGROUND: Rotavirus as a causative agent of childhood diarrhea is known to cause serious illness among children less than 5 years of age. This study examined the epidemiology of rotavirus disease burden and diversity of G and P genotypes of rotavirus in Nepal. METHODS: Stool samples were tested for rotavirus by Enzyme Immuno Assay and Group A rotaviruses were detected by using both ELISA and RT-PCR in 2718 samples between 2009 and 2011. RESULTS: Rotavirus was more frequently detected among inpatients (28.5%) than outpatients (15.2%). Over the three-year study period, 653 (24.4%) cases were positive for rotavirus by ELISA. Genotyping by RT-PCR was done on 638 samples. The most prevalent genotype was G12P [6] (60.4%). Mixed infections were not uncommon (14% in 2009, 29% in 2010 an 7% in 2011). However, 41 were partially typed and 23 were completely untyped over the study period. CONCLUSIONS: This study highlights the rotavirus disease burden and diversity of rotavirus strains circulating in Nepal. Continued sentinel surveillance will provide useful information to policy makers with regard to rotavirus vaccine introduction.