Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley.

This cross-sectional study was designed to identify information on the frequency, antimicrobial resistance and species diversity of methicillin-resistant coagulase negative staphylococci (MRCoNS) among pet dogs and humans within households. Fifty five nasal swabs each from dogs and their owners were collected. MRCoNS were identified based on gram staining, culture on mannitol salt agar, biochemical tests, and mecA gene amplification. The antibiotic susceptibility of the isolates was assessed by a disc diffusion test. Uniplex and multiplex polymerase chain reaction (PCR) were employed for the species identification of MRCoNS and SCCmec typing, respectively. Species were further confirmed by MALDI-TOF-MS. The prevalence of MRCoNS was 29% in dog owners and 23.6% in dogs. Four different species of MRCoNS, Staphylococci saprophyticus (48.3%), S. haemolyticus (24.1%), S. warneri (17.2%), and S. epidermidis (10.3%), were detected. Two isolates each from dog owners and dogs showed a constitutive resistance to macrolide-lincosamide-streptogramin B (cMLSB) resistance, eight isolates each from dogs and their owners showed a macrolide-streptogramin B (MSB) resistance, and only two isolates from dog owners revealed an inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB) resistance. SCCmec types were SCCmec type IV (55.2%), SCCmec type V (24.1%), SCCmec III (10.3%), SCCmec II (3.4%); two isolates were non-typable. MRCoNS are prevalent and genetically diverse in companion animals and humans. Different species of MRCoNS were found in dogs and their owners.

Staphylococcus aureus and Methicillin Resistant S. aureus in Nepalese Primates: Resistance to Antimicrobials, Virulence, and Genetic Lineages.

Staphylococcus aureus is a ubiquitous pathogen and colonizer in humans and animals. There are few studies on the molecular epidemiology of S. aureus in wild monkeys and apes. S. aureus carriage in rhesus macaques (Macaca mulatta) and Assam macaques (Macaca assamensis) is a species that has not previously been sampled and lives in remote environments with limited human contact. Forty Staphylococcus aureus isolates including 33 methicillin-susceptible S. aureus (MSSA) and seven methicillin-resistant S. aureus (MRSA) were characterized. Thirty-four isolates were from rhesus macaques and six isolates (five MSSA, one MRSA) were from Assam macaques. Isolates were characterized using StaphyType DNA microarrays. Five of the MRSA including one from Assam macaque were CC22 MRSA-IV (PVL+/tst+), which is a strain previously identified in Nepalese rhesus. One MRSA each were CC6 MRSA-IV and CC772 MRSA-V (PVL+). One MSSA each belonged to CC15, CC96, and CC2990. Six MRSA isolates carried the blaZ, while ten known CC isolates (seven MRSA, three MSSA) carried a variety of genes including aacA-aphD, aphA3, erm(C), mph(C), dfrA, msrA, and/or sat genes. The other 30 MSSA isolates belonged to 17 novel clonal complexes, carried no antibiotic resistance genes, lacked Panton-Valentine Leukocidin (PVL), and most examined exotoxin genes. Four clonal complexes carried egc enterotoxin genes, and four harbored edinB, which is an exfoliative toxin homologue.

The emergence of colistin-resistant Escherichia coli in chicken meats in Nepal.

The emergence and dissemination of colistin resistance among Gram-negative bacteria is a global problem. We initiated a surveillance of colistin-resistant and -susceptible Escherichia coli in raw meats from chicken in Nepal. A total of 180 meat samples were collected; from these, 60 E. coli strains were isolated (33.33%), of which 16 (26.66%) were colistin-resistant and harboured the mcr-1 gene. All isolates were characterised by antibiotic susceptibility testing, the presence of antibiotic resistance genes, phylogenetic analysis and plasmid replicon typing. Most of the colistin-resistant E. coli had the antibiotic resistant pattern CIP/CN/SXT/TE (43.75%). Coexistence of tet, qnr, sul and dfr genes was detected in both colistin-resistant and -susceptible E. coli. Most colistin-resistant E. coli strains belonged to phylogroup C, whereas 10% of isolates belonged to phylogroup D. Inc FIB was the dominant plasmid Inc type in the isolates. Dissemination of antibiotic-resistant E. coli in raw meats is a public health concern in Nepal and requires further investigation to ascertain the sources of contamination.

Molecular Characterization of Colistin-Resistant Escherichia coli Isolated from Chickens: First Report from Nepal.

Dissemination of mcr-1 encoding colistin resistance in Gram-negative bacteria has created critical situation in poultry, livestock farming, and public health. In Nepal, for the first time, we initiated surveillance of colistin-resistant Escherichia coli in broilers from seven different chicken farms. A total of 324 cloacal swabs were collected and 118 E. coli were isolated, of which 27 (22.8%) were colistin resistance all harboring mcr-1 gene, but lacking ISApI1. Colistin-resistant isolates were characterized by antibiotic susceptibility testing, detecting antibiotic resistance genes, phylogenetic analysis, and plasmid replicon typing. These isolates belonged to the phylo-group A (70.37%) and phylo-group D (29.63%). In addition, most isolates (>80%) were resistant to ciprofloxacin, tetracycline, and sulfamethoxazole-trimethoprim. As much as 3 of the 27 mcr-1 encoding isolates were confirmed as extended-spectrum ß-lactamase (ESBL) producer, all 3 isolates carrying blaCTX-M gene. We performed the conjugation experiment to check transferability of mcr-1, tet, and blaCTX-M genes, and only two donors were found to have transferred resistance to ticarcillin. The transfer of colistin and tetracycline resistance was not detected, which suggests the chromosomal location of mcr-1 and tet genes. The prevalence of Inc K/B and Inc I1 was 96.3% and 81.48%, respectively. This study shows the co-existence of mcr-1 with tet, sul, qnr, dfr, and blaCTX-M genes and dissemination of these resistant isolates in Nepalese chicken farms, which may pose huge threat to the livestock, especially chickens, and public health in Nepal.

MRSA Strains in Nepalese Rhesus Macaques (Macaca mulatta) and Their Environment.

This study looked at 227 saliva samples from Rhesus macaques (Macaca mulatta) and 218 samples from the surrounding environments. From these samples, MRSA isolates were collected from Rhesus saliva samples (n = 13) and environmental samples (n = 19) near temple areas in Kathmandu, Nepal. For comparison, selected MRSA isolates (n = 5) were obtained from patients with wound infections from a Kathmandu hospital. All isolates were characterized using Abbott StaphyType® DNA microarrays. Eighteen isolates (62%) from monkeys (n = 4; 31%) and environmental samples (n = 14; 74%), were CC22-MRSA-IV. Most (n = 16) of them carried both, the PVL locus and toxic shock toxin gene (tst1), an unusual combination which is the same as in previously characterized strain from Nepalese macaques and pigs. The five human isolates also belonged to that strain type. Eight monkey MRSA isolates were CC361-MRSA-IV. One MRSA from a monkey and one from an environmental sample, were CC88-MRSA-V. Other environmental MRSA included one each, CC121-MRSA-VT, and CC772 -MRSA-V. Two were CC779-MRSA-VT, potentially a novel clone. All MRSA carried the blaZ gene. The aacA-aphD, dfrA, and erm (C) genes were very common in isolates from all sources. One macaque MRSA carried the resistance genes aphA3 and sat, neither previously identified in primate MRSA isolates. This current study suggests that humans could be a potential source of the MRSA in the macaques/environment and transmission may be linked to humans feeding the primates and/or living in close proximity to each other.

The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: is man the common source?

Swine nasal samples [n = 282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n = 59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates.

Co-existence of blaOXA-23 and blaNDM-1 genes of Acinetobacter baumannii isolated from Nepal: antimicrobial resistance and clinical significance.

BACKGROUND: Molecular analysis of carbapenem-resistant genes in Acinetobacter baumannii, an emerging pathogen, is less commonly reported from Nepal. In this study we determined the antibiotic susceptibility profile and genetic mechanism of carbapenem resistance in clinical isolates of A. baumannii. METHODS: A. baumannii were isolated from various clinical specimens and identified based on Gram staining, biochemical tests, and PCR amplification of organism specific 16S rRNA and blaOXA-51 genes. The antibiotic susceptibility testing was performed using disc diffusion and E-test method. Multiplex PCR assays were used to detect the following ß-lactamase genes: four class D carbapenem hydrolyzing oxacillinases (blaOXA-51, blaOXA-23, blaOXA-24 and blaOXA-58). Uniplex PCRs were used to detect three class B metallo-ß-lactamases genes (blaIMP, blaVIM and blaNDM-1), class C cephalosporin resistance genes (blaADC), aminoglycoside resistance gene (aphA6), and ISAba1 of all isolates. Insertion sequence ISAba125 among NDM-1 positive strains was detected. Clonal relatedness of all isolates were analyzed using repetitive sequence-based PCR (rep-PCR). RESULTS: Of total 44 analyzed isolates, 97.7% (n = 43) were carbapenem-resistant A. baumannii (CR-AB) and 97.7% (n = 43) were multidrug resistant A. baumannii (MDR-AB). One isolate was detected to be extremely drug resistant A. baumannii (XDR-AB). All the isolates were fully susceptible to colistin (MICs < 2 µg/ml). The blaOXA-23 gene was detected in all isolates, while blaNDM-1 was detected in 6 isolates (13.6%). Insertion sequence, ISAba1 was detected in all of blaOXA-23 positive isolates. ISAba125 was detected in all blaNDM-1 positive strains. The blaADC and aphA6 genes were detected in 90.1 and 40.1%, respectively. The rep-PCR of all isolates represented 7 different genotypes. CONCLUSION: We found high prevalence of CR-AB and MDR-AB with blaOXA-23 gene in a tertiary care hospital in Nepal. Systemic network surveillance should be established for monitoring and controlling the spread of these resistant strains.

Detection of metallo-ß-lactamases-encoding genes among clinical isolates of Pseudomonas aeruginosa in a tertiary care hospital, Kathmandu, Nepal.

OBJECTIVES: This study was carried out to determine the prevalence of metallo-ß-lactamases (MBLs) producing Pseudomonas aeruginosa in imipenem-nonsusceptible isolates and to detect MBL-encoding genes among MBLs-positive isolates. RESULTS: Metallo-ß-lactamases production was detected in 68.6% isolates of P. aeruginosa with reduced susceptibility to imipenem. The bla VIM-2 gene was detected in 75% isolates and bla IMP-1 was detected in 25% isolates. All MBLs-positive isolates were multidrug resistant with a high level of resistance to imipenem (MIC 16 to ≥ 32 µg/ml), meropenem (MIC 16 to ≥ 32 µg/ml), and ceftazidime (MIC 64 to ≥ 512 µg/ml). All MBL-positive isolates were susceptible (MIC ≤ 2 µg/ml) to colistin. We found high prevalence of MBL-producing P. aeruginosa. To our knowledge this is the first report of detection of bla VIM-2 and bla IMP-1 in P. aeruginosa from Nepal. This indicates the need for awareness to prevent the spreading of these resistant isolates in hospital setting.

Design and development of a prototype 25 kV, 10 A long pulse Marx modulator for high power klystron.

Research, design, and development of high average power modulators for the proposed Indian Spallation Neutron Source are in progress at Raja Ramanna Centre for Advanced Technology. With this objective, a prototype of 25 kV, 10 A, 1 ms Marx modulator at repetition rate of 1 Hz has been designed and developed which serves as a proof of principle and technology assessment stage for further development of high repetition rate high voltage high average power modulators. Insulated Gate Bipolar Transistor (IGBT) based modules of 2.8 kV switching capability have been used as main modules. The modulator had 8.2% droop in output voltage pulse without any droop compensation circuit. A droop compensation involving 15 corrector modules has been used to reduce the droop up to 1%. We have used IGBT based 250 V switches to realize the corrector module. A microcontroller based control unit was designed and developed for triggering the main and corrector modules. With this control unit, programmable output pulse has been achieved. Electrical isolation between high voltage circuits and control circuit has been achieved by the use of fiber optic based control signal transmission. Output pulses of 1 ms pulse width, 800 ns rise time, and 5 µs fall time have been achieved. The modulator has advantages of modular design, adjustable pulse width, adjustable rise time, and fall time.

Emergence of staphylococcal cassette chromosome mec type Ⅰ with high-level mupirocin resistance among methicillin-resistant Staphylococcus aureus

Objective: To investigate the molecular epidemiology and antimicrobial resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) among healthcare workers and patients. Methods: MRSA isolates were recovered from nasal swabs collected at a tertiary care hospital of Nepal and confirmed on the basis of Gram staining, conventional biochemical tests, and PCR amplification of mecA gene. PCRs were also used for detection of the different resistance genes and staphylococcal cassette chromosome (SCC) mec types. Antibiotic susceptibility patterns of isolates were assessed by disc diffusion method and minimum inhibitory concentrations were determined by E-test. Results: A total of 29 MRSA were isolated from 536 nasal swabs (5.4%) of health care workers and patients at a tertiary care hospital in Nepal. All isolates were susceptible to amikacin, gentamicin, vancomycin (minimal inhibitory concentrations1024μg/mL). Fourteen isolates were found harboring the mupA gene and one isolate was found carrying the novel mupB gene. High prevalence (68%) of SCCmec I type was found, followed by SCCmec V (13%) and SCCmec III (3%) among all the MRSA isolates. Conclusions: We found the emergence of SCCmec type Ⅰ with high-level mupirocin resistance among MRSA in Nepal. Data also suggest that MRSA SCCmec type V strain has spread from the community to the hospital.