Molecular detection of enterotoxins in multidrug resistant Aeromonas from ready to eat foods in North Western Himalayas: Public health significance.

Aeromonas spp. are normal inhabitants of aquatic environments and are emerging foodborne bacterial pathogens. Aeromonas spp. contamination is frequent in ready-to-eat (RTE) seafood and can also occur in products prepared from milk or meat. The study determined the enterotoxin and antimicrobial susceptibility profiles of Aeromonas spp. isolates recovered from RTE milk products (n = 105), RTE meat/fish products (n = 40) and drinking water (n = 60) samples collected from tourist places in Himachal Pradesh, India, in northwestern Himalayas. 7.3 % (16/220) samples were found contaminated with Aeromonas spp. These isolates were identified as A. hydrophila (31.3 %), A. schubertii (25.0 %), A. sobria (25.0 %) and A. veronii (18.8 %). Aeromonas spp. contamination was significantly higher (14.3 %, 15/105, p = 0.0001) in RTE milk products. The contamination levels for water samples were 1.7 % whereas none of the tested RTE meat or fish products yielded Aeromonas spp. Among RTE milk products, contamination was significantly higher in paneer (South Asian soft cheese) (26.1 %, p = 0.0027) and cream (25.0 %, p = 0.046) based RTE foods. All isolates carried alt (361 bp), encoding a cytotonic heat-labile enterotoxin. Ampicillin resistance was 100 % and high levels (>30 %) of resistance were recorded for amoxicillin/clavulanic acid, amikacin, cefotaxime and ceftazidime. Six (37.5 %) isolates were multi drug resistant (MDR), showing resistance to aminoglycosides, cephams and penicillins. Isolation of alt carrying MDR isolates from RTE foods indicates that Aeromonas spp. can be potential foodborne public health threat in northwestern Himalayas.

Prevalence and phylogeography of Taenia hydatigena metacestodes from goats of India.

The study determined the prevalence and genetic population structure relationships of Cysticercus tenuicollis (Taenia hydatigena metacestode) retrieved from the goats slaughtered in north India. An overall prevalence of 9.62% (59/613) was recorded. Genetic population structure relationships were assessed by targeting partial cytochrome c oxidase subunit 1 mitochondrial gene sequence. Phylogenetic tree analysis revealed that all the present study representative isolates (n = 7) formed a major clade and grouped with T. hydatigena isolates retrieved from sheep, goats, pigs and dogs, originating from China, Iran, Nigeria, Ghana and Poland. However, a single isolate from Himachal Pradesh (isolate 3) formed a subgroup within the clade. The neutrality and diversity indices revealed high values of haplotype diversity [Hd = 0.99695 (0.95238­1.0000)] and low nucleotide diversity (π = 0.49276), which was indicative of demographic expansion and low gene flow, suggesting that Indian T. hydatigena isolates were not genetically differentiated. Tajima's D (−1.26988) and Fu and Li's D statistics values (−0.74556) were negative, demonstrating deviations from neutrality and both propounded recent population expansion or purifying selection. Results highlighted a low genetic diversity of T. hydatigena metacestodes across the geographical range of north India.

Biodiversity of meatborne Listeria spp. in Himachal Pradesh and their interaction with indigenous probiotics.

This study determined the anti-listerial activity of indigenous probiotics from traditional fermented foods of Western Himalaya against meat borne Listera monocytogens isolates from Himachal Pradesh. One hundred samples of meat and meat products like chicken (n = 25), chevon (goat meat, n = 20), fish (n = 20) and pork (n = 30) were collected and were analyzed for the presence of Listeria spp. by recommended culture and biochemical methods. L. monocytogens isolates were confirmed by PCR targeting the virulence gene hlyA (haemolysin A) and by16S rRNA sequencing. Anti-listerial activity of probiotic bacteria isolated from indigenous fermented foods of Himachal Pradesh was determined by well diffusion method using Lactobacillus rhamnosus GG (ATCC 53103) as the reference strain. Five percent of tested samples were found positive for L. monocytogens with incidence of 8.0% in chicken (2/25), 10.0% in fish (2/20) and 4.0% in chevon meat (1/25). None of the tested pork samples were found contaminated with L. monocytogenes. Among 11 indigenous probiotics used in this study, highest antagonistic activity was exhibited by Lactobacillus plantarum (ADF 10) and Enterococcus faecium (ADF1) which was equivalent to the reference strain.

Bacillus cereus: public health burden associated with ready-to-eat foods in Himachal Pradesh, India.

The study determined incidence, enterotoxigenecity and antimicrobial susceptibility profiles of Bacillus cereus isolated from ready-to-eat (RTE) milk products (n = 80), RTE meat products (n = 40), beverages (n = 40) and water samples (n = 60, from food preparing and serving outlets/restaurants) collected from eight different tourist places of Himachal Pradesh. 11.4% (25/220) samples were contaminated with Bacillus and isolates were identified as B. cereus (76.0%, n = 19), B. alvei (12.0%, n = 3), B. polymyxa (8.0%, n = 2) and B. firmus (4.0%, n = 1) by conventional and molecular methods. B. cereus incidence was highest in cheese based foods (25.0%) followed by vegetable soups (16.7%), khoa based foods (14.0%), milk based beverages (10.5%), paneer based foods (8.6%), cream based foods (8.3%) and water (8.3%) samples. Multiplex polymerase chain reaction detected enterotoxigenic genes only in B. cereus isolates. nhe complex (encoding non-haemolytic enterotoxins, ABC) genes were detected only in B. cereus isolates. 57.6% (11/19), 36.8% (7/19) and 5.3% (1/19) harboured all three (nheA, nheB, nheC), two (nheB, nheC) and one (nheC) nhe gene, respectively. Among hbl complex genes (encoding haemolytic enterotoxins CAD), only hblC (36.8%, 7/19) was detected. Incidence B. cereus cytK (encoding cytotoxin enterotoxin) was 52.6% (10/19). Each B. cereus isolate harboured two or more enterotoxigenic genes. Seven isolates had at least one gene from haemolytic and non-haemolytic complexes along with cytK. High levels (> 50%) of antimicrobial resistance were recorded for penicillin, amoxicillin, ampicillin cefixime and ceftazidine in tested B. cereus isolates. Two isolates were identified as multidrug resistant isolates with resistance to ≥ 3 antibiotic classes.

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5'-carboxyfluorosceinsuccidyl ester.

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells. RESULTS: We observed that 20.3% (+/- 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner. CONCLUSIONS: Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.

Incidence and antimicrobial susceptibility of Staphylococcus aureus isolated from ready-to-eat foods of animal origin from tourist destinations of North-western Himalayas, Himachal Pradesh, India.

This study was aimed to determine the incidence of Staphylococcus aureus in ready-to-eat (RTE) milk (n = 120) and meat (n = 120) products from various tourist places in north western Himalayas, Himachal Pradesh, India. S. aureus isolates and its enterotoxins; A, B, D and E were characterized by conventional and molecular methods. Antimicrobial susceptibility (AMS) profiles of S. aureus isolates were determined by disk diffusion method using Clinical and Laboratory Standards Institute criteria. Overall, 6.7% (n = 16/240) food samples were positive for S. aureus. PCR amplification of nucA confirmed all biochemically characterized isolates as S. aureus. Incidence of S. aureus was higher (10.0%) in RTE milk products than meat products (3.3%). S. aureus contamination levels were highest in milk cake/khoa (26.0%, p = 0.0002) followed by ice cream/kulfi (10.0%, p = 0.4), mutton momo (10.0%, p = 0.4), burfi (3.3%, p = 0.7) and chicken momo (3.3%, p = 0.7). None of the isolates carried genes for S. aureus enterotoxins; A, B, D and E. AMS testing revealed seven different resistance patterns and 81.3% multi drug resistance. All the isolates were resistant to ampicillin. High resistance levels were observed against methicillin (93.7%), clindamycin (68.8%), erythromycin (56.3%) and vancomycin (43.8%). Vancomycin resistant (n = 7) isolates were also resistant to methicillin. All isolates were susceptible to novobiocin.

Quality assurance for antimicrobial susceptibility testing of Neisseria gonorrhoeae in Latin American and Caribbean countries, 2013-2015.

OBJECTIVES: A Neisseria gonorrhoeae antimicrobial susceptibility quality control comparison programme was re-established in Latin America and the Caribbean to ensure antimicrobial susceptibility data produced from the region are comparable nationally and internationally. METHODS: Three panels, consisting of N. gonorrhoeae isolates comprising reference strains and other characterised isolates were sent to 11 participating laboratories between 2013 and 2015. Antimicrobial susceptibilities for these isolates were determined using agar dilution, Etest or disc diffusion methods. Modal minimum inhibitory concentrations (MICs) for each panel isolate/antibiotic combination were calculated. The guidelines of the Clinical and Laboratory Standards Institute were used for interpretations of antimicrobial susceptibility. The agreement of MICs with the modal MICs was determined for each of the participating laboratories as well as for each of the antibiotics tested. RESULTS: Five of 11 laboratories that participated in at least one panel had an overall average agreement between participants' MIC results and modal MICs of >90%. For other laboratories, agreements ranged from 60.0% to 82.4%. The proportion of agreement between interpretations for all the antibiotics, except penicillin and tetracycline, was >90%. The percentages of agreement between MIC results and their modes for erythromycin, spectinomycin, cefixime and azithromycin were >90%. Tetracycline, ceftriaxone and ciprofloxacin agreement ranged from 84.5% to 89.1%, while penicillin had 78.8% agreement between MICs and modal MICs. CONCLUSIONS: The participating laboratories had acceptable results, similar to other international quality assurance programmes. It is important to ensure continuation of the International Gonococcal Antimicrobial Susceptibility Quality Control Comparison Programme to ensure that participants can identify and correct any problems in antimicrobial susceptibility testing for N. gonorrhoeae as they arise and continue to generate reproducible and reliable data.

Resistance to Ceftriaxone and Azithromycin in Neisseria gonorrhoeae Isolates From 7 Countries of South America and the Caribbean: 2010-2011.

Seven countries in Latin America and the Caribbean report on (2010 and 2011) the susceptibility of 2235 isolates of Neisseria gonorrhoeae to 6 antibiotics. Thirteen isolates had ceftriaxone minimum inhibitory concentrations (MICs) of 0.125 to ≥ 0.25 mg/L. The percentage of resistant isolates to the following antibiotics was: azithromycin, 1.0% to 1.7%; ciprofloxacin, 42.1% to 36.2%; penicillin, 31% to 35%; tetracycline, 21.8% to 22.6%.

Two decades of the gonococcal antimicrobial surveillance program in South America and the Caribbean: challenges and opportunities.

OBJECTIVES: The WHO has called for a global action plan to control the spread and impact of antibiotic resistant Neisseria gonorrhoeae. We report on key antimicrobial susceptibility (AMS) trends in countries in Latin America and the Caribbean from 1990 to 2011. METHODS: Data collected between 1990 and 2011 by up to 23 countries in the Latin American and Caribbean region were aggregated and analysed for overall trends in N gonorrhoeae AMS to six antibiotics. Methods for gonococcal identification, susceptibility testing and interpretation were standardised. RESULTS: More than 21 500 N gonorrhoeae isolates were tested for AMS between 1990 and 2011. The number of countries reporting yearly declined from 17 in the 1990 s to 7 in 2011. The first isolates (0.4%, 20/5171) with reduced susceptibility (minimum inhibitory concentration ≥ 0.125 mg/L) to ceftriaxone were reported between 2007 and 2011. Ciprofloxacin resistance, first noted in the mid-1990 s, ranged from 1.6% of isolates tested in 1997 rising to 42.1% in 2010. Overall, azithromycin resistance reached a high of 25.8% of isolates tested in 2008 falling to 1.0% in 2010. Resistance to penicillin ranged between 24.2% in 2003 to a high of 48.5% in 1993. Tetracycline resistance ranged between a high of 61.1% of isolates tested in 2001 to 21.8% in 2010. Plasmid mediated penicillin and tetracycline resistance declined over the period. CONCLUSIONS: Gonococcal AMS surveillance initiatives are urgently needed in every country in the region to ensure that effective treatments for gonococcal infections are in place and to better understand emerging trends in gonococcal antimicrobial resistance.