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DETECTION OF VIRULENCE GENE PROFILE AND ESBL PRODUCTION IN Shigella spp. FROM FOODS OF ANIMAL ORIGIN
(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) SRAVYA DEEPIKA GUNTI; SUBHASHINI .N (MAJOR); BINDU KIRANMAYI .CH; SUDHAKAR .K
The present study was undertaken for isolation and characterization of Shigella spp. from foods of animal origin. A total of 367 samples from wide range of foods of animal origin comprising of chicken (70), mutton (80), pork (50), fish (65), raw milk samples (65), spoiled eggs contents (12) and fresh eggs contents (25). Out of 367 samples analyzed, 15.2% (56/367) and 1.3% (5/367) samples were positive for Shigella spp. by cultural isolation and m-PCR, respectively. All the five S. dysenteriae isolates were detected from egg samples. None of the isolates exhibited biofilm production ability on congo red agar and 60% Shigella isolates showed lipase activity with clear zone around the colonies on Tween20 agar. All the isolates carried the virulence gene virA (100%) followed by sat (80%), set1A (60%) and Ial and set1B (40% each) whereas stx and sen genes were not found in any of the Shigella spp. Antibiogram of five isolates revealed 100% resistance towards Penicillin-G (100%), followed by ampicillin and amikacin (80% each), tetracycline and streptomycin (60% each), cefoxitin, azithromycin, chlormphenicol and nalidixic acid (40% each) and gentamicin, ciprofloxacin and nitrofurantoin (20% each). Higher sensitivity was observed towards ciprofloxacin (80%), followed by nalidixic acid (60%), tetracycline, nitrofurantoin, chlormphenicol and azithromycin (40% each) and gentamicin, streptomycin and ampicillin (20% each). All five S. dysenteriae isolates (100%) isolates were found to be multi drug resistant (MDR). A total of two (40%) isolates were phenotypically confirmed as ESBL producers and none of the isolates showed any of the β-lactamase genes under study. ERIC-PCR and REP-PCR genotyping distinguished five isolates of S. dysenteriae into five genotypes revealed a greater degree of heterogeneity. The discriminatory power of ERIC-PCR and REP-PCR was found to be one, indicating that both the genotyping methods were highly significant. Cluster analysis also revealed a great degree of homogeneity and heterogeneity among different isolates recovered from different sources, thereby indicating possible chance of interactions among different environments.
Open Access
ANTIMICROBIAL RESISTANCE, VIRULENCE AND GENOTYPIC PROFILING OF Clostridium difficile ISOLATED FROM ANIMALS AND HUMANS
(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) NAGARJUNA REDDY NAKKA; SRINIVASA RAO .T (MAJOR); BINDU KIRANMAYI .CH; SUDHA RANI CHOWDARY .CH
The present study was undertaken for isolation and characterization of Clostridium difficile from food animals, foods of animal origin, environmental samples and human samples. Out of 400 samples analyzed, 15% (60/400) were positive for C. difficile by cultural isolation and confirmation by biochemical tests. Out of 60 phenotypically positive isolates from different sources, 11 (18.33%) isolates were further confirmed by species specific PCR targeting genes (tpi and gluD genes) The highest rate of occurrence of C. difficile was obtained in chicken samples 8.0% (4/50), while lowest in mutton samples 2% (1/50). All the C. difficile isolates (100%) showed gelatinase activity, 45.45% isolates exhibited congo red binding activity, none of the isolates were positive for lecithinase activity on on egg yolk agar. Eight C. difficile isolates (72.72%) carried both toxin A and toxin B, two (18.18%) isolates carried only toxin B, 10 (90.91%) isolates carried binary toxin and one (9.09%) isolate from diarrhoeic stools of vety students did not carry any of the toxins. Among 11 C. difficile isolates higher resistance was observed towards pencillin G and linezolid (100% each), followed by gentamicin (81.81%), cefotaxime and clindamycin (72.72% each) and ciprofloxacin (63.63%). The highest sensitivity was observed for chloramphenicol (100%) followed by metronidazole and vancomycin (81.81% each), moxifloxacin (72.72%) and meropenem (54.54%). Intermediate resistance pattern were observed against ceftriaxone and tetracycline (18.18% each). All the C. difficile isolates were MDR and MAR indexing of all isolates yielded 6 MAR index groups. Out of 11 C. difficile isolates nine were identified as ESBL suspects by PST and ESBL production was confirmed in nine (81.81%) isolates by CDM and in eight (72.72%) isolates by DDST. All the 9 phenotypic ESBL suspects did not carry any of the beta lactamase genes (blaTEM, blaSHV and blaOXA) by multiplex PCR. Among 11 C. difficile isolates, three (27.27%) isolates carried tetM genes, two (18.18%) isolates carried vanC1 gene and none of the isolates carried aac-aph genes by PCR. A greater degree of heterogeneity was observed among 11 C. difficile isolates from different sources by REP-PCR. Genotyping of C. difficile by REP-PCR was highly significant since the discriminatory power is one. Cluster analysis also revealed a greater degree of homogeneity and heterogeneity among different isolates recovered from different sources.
Open Access
MOLECULAR CHARACTERIZATION OF Clostridium perfringens ISOLATED FROM ANIMALS AND HUMANS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-03) SHARMILA GELLANKI; SRINIVASA RAO .T (MAJOR); SUBHASHINI .N; SUDHAKAR .K
The present study was undertaken for isolation and characterization of C. perfringens from animals, foods of animal origin and human samples. Out of 395 samples analyzed, 22.7% (90/395) were positive for C. perfringens by cultural isolation and confirmation by biochemical tests. Out of 90 phenotypically positive C. perfringens isolates from different sources, 34 (37.7%) isolates were further confirmed by species specific PCR. The highest rate of occurrence of C. perfringens was detected in mutton samples (18.1%) while lowest in human stool swab (5%) samples. All the C. perfringens isolates (100%) showed lecithinase activity on egg yolk agar, 82.3% isolates exhibited congo red binding activity, 73.5% isolates showed gelatinase activity and 88.2% isolates showed hemolytic activity on sheep blood agar plates. Eight (23.5%) C. perfringens isolates carried cpa gene indicating toxinotype A, four (11.7%) isolates harboured both cpa and cpe genes indicating toxinotype F and three (8.8%) isolates carried cpe gene alone. None of the isolates did not carry other three virulent genes like etx, cpb and cpi. Among 34 C. perfringens isolates higher resistance was observed towards erythromycin, linezolid, metronidazole, pencillin G, rifampicin (100%), followed by gentamicin (91.1%), tetracycline (88.2%), teicoplanin (85.2%), ciprofloxacin (73.5%) and chloramphenicol (32.3%). The sensitivity percentages were highest against imipenem (88.2%) followed by ceftriaxone (14.7%). Intermediate resistant patterns were observed against ceftriaxone (85.2%), chloramphenicol (67.6%) and ciprofloxacin (26.4%). All the 34 C. perfringens isolates were MDR and MAR indexing of all isolates yielded 6 MAR index groups. Out of 34 C. perfringens isolates 19 were identified as ESBL suspects by PST and ESBL production was confirmed in ten isolates by CDM and in four isolates by DDST. All the 19 phenotypic ESBL suspects did not carry any of the beta-lactamase genes (blaTEM, blaSHV and blaOXA) by multiplex PCR. A greater degree of heterogeneity was observed among 34 C. perfringens isolates from different sources by ERIC and REP-PCR. ERIC-PCR distinguished 33 genotypes and REP-PCR distinguished 34 genotypes of C. perfringens isolates. Genotyping of C. perfringens by ERIC-PCR and REP-PCR was highly significant since the discriminatory power is >0.9 (0.998 for ERIC–PCR and 1 for REP-PCR). Cluster analysis also revealed a greater degree of homogeneity and heterogeneity among different isolates recovered from different sources indicating the chances of cross contamination.
Open Access
MOLECULAR STUDIES ON Acinetobacter baumannii ISOLATES OF ANIMAL AND HUMAN ORIGIN
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-03) RESHMA MARADANA; SRINIVASA RAO .T (MAJOR); BINDU KIRANMAYI .CH; ASWANI KUMAR .K
The present study was undertaken for isolation and characterization of Acinetobacter baumannii from foods of animal origin, rectal/cloacal swabs of animals/ birds and human clinical samples. Out of 420 samples analyzed, 21 (5%) samples were positive for A. baumannii by m-PCR. About 12% of human blood samples, 10% of milk samples, 6.6% of chicken samples, 4% of human urine samples and 2.8% of sheep rectal swabs samples were positive for A. baumannii. Fifty two percent of A. baumannii isolates showed gelatinase activity on gelatin agar and none of the isolates exhibited biofilm production ability on congo red agar. About 28.57% of A. baumannii isolates carried the virulence genes csgA gene and 14% of the isolates harboured fimH gene whereas cnf1 and afa/draBC genes were not found in any of the A. baumannii isolates. Antibiogram of 21 A. baumannii isolates revealed 100% resistance towards penicillin-G and polymyxin B, followed by co-trimoxazole 90.4%, gentamicin 61.9%, amikacin 47.6%, cefepime and colistin (42.8%). Higher sensitivity was observed for ciprofloxacin and tigecycline (100%), followed by tetracycline (80.9%), imipenem (71.4%) and meropenem (66.6%). Colistin resistance genes mcr-1 and mcr-2 were detected in two isolates of human blood samples out of nine isolates which were phenotypically resistant to colistin. A total of three (14.3%) human isolates were phenotypically confirmed as ESBL producers and blaOXA was the only β- lactamase gene detected in two A. baumannii isolates of human blood samples. ERIC-PCR genotyping distinguished 19 genotypes among 21 isolates of A. baumannii. REP-PCR genotyping distinguished 20 genotypes among 21 A. baumannii isolates. ERIC-PCR and REP-PCR analysis revealed a greater degree of heterogeneity among 21 A. baumannii isolates from different sources. The discriminatory power of ERIC-PCR and REP-PCR was found to be 0.990 and 0.995, respectively indicating both the genotyping methods were highly significant. Cluster analysis also revealed a great degree of homogeneity and heterogeneity among different isolates recovered from different sources thereby indicating that possible chance of interactions among different environments.
Open Access
DETECTION OF ANTIBIOTIC RESISTANCE, GENETIC DIVERSITY IN Klebsiella pneumoniae ISOLATED FROM FOODS OF ANIMAL ORIGIN AND ITS PUBLIC HEALTH SIGNIFICANCE
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-03) VENKATALAXMI ADIMULAM; SUBHASHINI .N (MAJOR); SRINIVASA RAO .T; ASWANI KUMAR .K
The present study was undertaken to characterize K. pneumoniae in foods of animal origin and human clinical samples based on cultural and molecular techniques. A total of 402 samples comprising different foods like milk, meat, egg and fish, human clinical samples such as blood, urine and nasal swabs, rectal/cloacal swabs of pigs and chickens were analyzed for the presence of K. pneumoniae. The overall occurrence of K. pneumoniae was found to be 6.47% (26/402) by cultural and molecular methods in samples obtained from different sources. Out of 26 K. pneumoniae isolates, highest occurrence was found in urine samples (7/26, 15.21%) followed by pork (6/44, 13.64%), eggs (2/15, 13.33%), milk (5/58, 8.62%), chicken (2/42, 4.76%) and blood (4/102, 3.92%) samples. Phenotypic virulence factors such as capsule, hypermucoviscosity and biofilm formation were detected in 26 (100%), 11(42.31%) and eight (30.77%) isolates, respectively whereas virulence genes such as uge, rmpA and Kfu were observed in eight (30.77%), four (15.38%) and three (11.54%) isolates, respectively. None of the isolates carried the aerobactin gene. Antibiogram profiling of K. pneumoniae isolates indicated the highest resistance to erythromycin, vancomycin and ampicillin in 100%, 100% and 92.3%, respectively followed by cefpodoxime (80.77%), amoxicillin-clavulanic acid (76.92%), cefixime (57.69%), ceftriaxone (34.61%). All the isolates were sensitive to imipenem followed by piperacillin-tazobactam (84.61%). Intermediate resistance was observed to colistin (69.23%). All K. pneumoniae isolates were found to be MDR and MAR index value ranged between 0.18 to 0.65. ESBL production was confirmed in 14 K. pneumoniae isolates by both phenotypic and molecular methods and blaOXA, was the predominant gene which was found in eight among 14 isolates, blaCTX-M1 and blaCTX-M9 were found in four and two isolates, respectively and the β-lactamase gene blaAmpC was detected only in four isolates. A greater degree of heterogeneity was observed among 14 ESBL-positive K. pneumoniae isolated from different sources as revealed by presence of 14 genotypes each by ERIC and REP-PCR analysis. All the 14 different K. pneumoniae subtypes were differentiated by ERIC-PCR and REP-PCR. Genotyping of ESBL-producing K. pneumoniae by ERIC-PCR and REP-PCR was found to be highly significant since the discriminatory power >0.9 is considered highly significant (one for both ERIC-PCR and REP-PCR). Cluster analysis also revealed a great degree of heterogeneity and also the homogeneity among different isolates recovered from different sources thereby indicating that there is a chance of cross-contamination between various foods of animal origin.
Open Access
ISOLATION AND MOLECULAR CHARACTERIZATION OF Bacillus cereus ISOLATED FROM MILK AND MILK PRODUCTS
(2023-04) KUPPILI, PRIYANKA; Ch, BINDU KIRANMAYI; T, SRINIVASA RAO; K, ASWANI KUMAR
The present study was undertaken to characterize the incidence, enterotoxigenic gene profile, antibiogram profile and genetic diversity of Bacillus cereus isolated from milk and different milk products. A total of 320 samples comprising of 150 raw milk and 170 milk product samples were analyzed for the presence of B. cereus. Overall prevalence of B. cereus in milk and milk products samples was found to be 9.68% (31/320) by species specific PCR targeting gyrB gene with 8% (12/150), 15% (3/20), 12.1% (4/33), 11.8% (2/17), 10.8% (4/37) and 21.4% (6/28) from raw milk, basundi, burfi, colostrum milk pudding (junnu), kalakhand and khoa samples, respectively. Out of 31 genotypically positive B. cereus isolates, 23 (74.1%) isolates were carrying atleast one of the virulence genes with nheA in 10 isolates (32.3%), nheB in 12 isolates (38.7%), nheC in 16 (51.6%) isolates and entFM in 14 isolates (45.2%) and none of the isolates were positive for hblA, hblC, hblD and cytK genes. Antibiogram profiling of 31 B. cereus isolates revealed that all the isolates (100%) were resistant to pencillin and amoxicillin-clavulanic acid followed by cefepime (87.1%), clindamycin (32.31%), rifampicin (29.0%), vancomycin (25.8%), colistin (19.4%) ciprofloxacin, chloramphenicol and erythromycin (6.5%) each and none of the isolates were resistant to gentamicin and tetracycline. None of the B. cereus isolates were positive for any of the ESBL genes (bla TEM, bla SHV, blaOXA and bla CTX-M). A greater degree of heterogeneity among the genotypically positive B. cereus isolates isolated from milk and different milk products was revealed by the presence of 28 genotypes by ERIC-PCR and 29 genotypes by REP-PCR analysis. Genotyping of B. cereus by ERIC-PCR and REP-PCR were found to be highly suitable since the discriminatory power above 0.9 are considered as highly significant (0.997 for ERIC PCR and 0.995 for REP-PCR). As the discriminatory power of ERIC-PCR is more than the REP-PCR, ERIC-PCR was able to differentiate the isolates better than REP-PCR. Cluster analysis using dendrograms revealed molecular heterogeneity and varying homogeneity among different isolates recovered from varied sources
Open Access
IDENTIFICATION AND CHARACTERIZATION OF MOTILE AEROMONADS ISOLATED FROM FISH AND FISH POND WATER WITH SPECIAL EMPHASIS ON VIRULENT Aeromonas hydrophila
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) NARENDRA KUMAR, GONEPUDI; SRINIVASA RAO, T (MAJOR); BINDU KIRANMAYI, Ch; ASWANI KUMAR, K
Motile aeromonads are one of the troublesome bacteria affecting the aquaculture industry by causing an assortment of diseases and also implicated in causing illness to humans by consumption of raw or improperly cooked fish, drinking contaminated water and contamination of wounds by water having aeromonads etc. The present study was intended to characterize Aeromonas spp. of fish, fish pond water and also human diarrhoeal origin with special emphasis to isolate and identify Virulent Aeromonas hydrophila (VAh) based on cultural and molecular techniques. A total of 330 samples comprising healthy and diseased fish, fish pond water and human diarrhoeal samples were analyzed for presence of Aeromonas spp. viz. A. veronii and A. hydrophila. Overall prevalence of genus Aeromonas was found to be 37.27% (123/330). Out of 123 Aeromonas isolates, m-PCR revealed 67 (54.47%) were to be A. veronii and seven (5.69%) were to be A. hydrophila. Out of 74 positive Aeromonas spp. isolated from different sources, highest prevalence of 56.75% (42/74) was found in healthy fish followed by diseased fish 25.67 (19/74), fish pond water 16.21% (12/74) and human diarrhoea 1.35% (1/74). Out of seven A. hydrophila isolates, three isolates were phenotypically positive for VAh based on inositol fermentation on M9I agar. Among 74 Aeromonas spp. isolates, the phenotypic virulence factors i.e. biofilm formation, gelatinase activity, DNase activity, caseinase activity, lipase activity and hemolysis were detected in 61(82.43%), 70 (94.59%), 69 (93.24%), 52 (70.27%), 48 (64.86) and 70 (94.59%) of the isolates, respectively. Out of 74 Aeromonas spp. isolates, 49 were positive for atleast one of the virulence genes. 49 Aeromonas spp. revealed the presence of virulence genes i.e act, ast, alt, ahyB, fla, lip, aer, ser, gcat and exu which were detected in 35 (47.29%), 9 (12.16%), 14 (18.91%), 15 (20.27%), 3 (4.05%), 26 (35.13), 16 (21.62%), 7 (9.45%), 16 (21.62%) and 12 (16.21%) of the isolates, respectively. Antibiogram profiling of 74 Aeromonas isolates unveiled a significant fraction of the Aeromonas isolates to be resistant towards ampicillin and novobiocin (100%) followed by vancomycin (71.62%) and erythromycin (54.05%). ESBL production was confirmed in 24 Aeromonas spp. isolates by both phenotypic and molecular methods and blaTEM was the predominant gene in 23 isolates and blaOXA gene was detected in only one isolate. A greater degree of heterogeneity was observed among 24 ESBL positive Aeromonas spp. isolated from different sources as revealed by presence of 24 genotypes each by ERIC and REP-PCR analysis. 19 different A. veronii and five A. hydrophila subtypes were differentiated by ERIC-PCR and REP-PCR. Genotyping of Aeromonas spp. by ERIC-PCR and REP-PCR was found to be highly significant since the discriminatory power >0.9 is considered as highly significant (1 for both ERIC –PCR and REP-PCR). Cluster analysis also revealed a great degree of homogeneity and heterogeneity among different isolates recovered from different sources thereby indicating that there is a chance of cross-contamination particularly in the fish markets.
Open Access
ISOLATION AND CHARACTERIZATION OF VIBRIO SPECIES FROM FISH AND FISH POND WATER
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) SUSMITHA, BUJUTI; Bindu Kiranmayi, CH (MAJOR); Srinivasa Rao, T; Aswani kumar, K
The consumption of raw, improperly cooked or cross-contaminated fish and fish products by Vibrio spp. can cause foodborne diseases. The present study was undertaken to characterize Vibrio species of fish and fish pond water origin based on cultural isolation. It was also aimed to determine the major antibiotic resistant genotypes and virulence genes of Vibrio species of worldwide public health importance. A total of 300 samples comprising fish and fish pond water samples were analyzed for presence of Vibrio spp. viz., V. parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus. Overall prevalence of genus Vibrio was found to be 55% (165/300). Out of 165 Vibrio isolates, m-PCR revealed 28 (16.9%) to be V. parahaemolyticus, 17(10.3%) to be V. alginolyticus and 15 (9.09%) to be V. cholerae and 11 (6.66%) to be V. vulnficus. Out of 131 positive Vibrio spp. in fish, highest prevalance was found in Catla catla (54.28%) followed by Labeo rohita (53.75%), Channa striata (48.38%) and other fishes (37.20%). Virulence genes are major indicators to pathogenicity of these microorganisms. Virulence genes ctxAB, tcp, rfbO1, zot were not detected in any of the V. cholerae isolates. Out of 28 V. parahaemolyticus isolates, virulence genes trh and tdh were detected in 21.42% and 10.71% of isolates, respectively. Out of 17 V. alginolyticus isolates, one (5.88%) isolate was carrying tdh gene. Out of 11 V. vulnificus isolates, cps allele 2 and vcgE were detected in 27.27% and 18.18% respectively and 18.18% of isolates were positive for both cps allelle 2 and vcg E. Antibiogram profiling of 71 isolates revealed a major fraction of the Vibrio isolates to be resistant towards ampicillin (100%) followed by amoxicillin/clavulanic acid (91.54%) and erythromycin (52.11%). ESBL production was confirmed in 15 Vibrio spp. isolates by both phenotypic and molecular methods and blaTEM was the predominant gene in 14 isolates and blaOXA gene was detected in only one isolate. A greater degree of heterogeneity was observed among 71 Vibrio isolates of different species from different sources as revealed by presence of 70 genotypes and 71 genotypes by ERIC and REP-PCR analysis, respectively. Twenty seven different V. parahaemolyticus, 17 V. alginolyticus, 15 V. cholerae and 11 V. vulnificus subtypes were differentiated by ERIC-PCR, whereas 28 different V. parahaemolyticus, 17 V. alginolyticus, 15 V. cholerae and 11 V. vulnificus subtypes by REP- PCR. Genotyping of Vibrio species by ERIC- PCR and REP- PCR was found to be highly significant since discriminatory power >0.9 is considered highly significant (0.9974 for ERIC PCR and 1 for REP-PCR. Cluster analysis also revealed a great degree of homogeneity and heterogeneity among different isolates recovered from different sources and indicating that there is a chance of cross-contamination particularly in the fish markets

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