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ThesisItem Open Access STUDIES ON MOLECULAR EPIDEMIOLOGY OF BRUCELLOSIS AND TUBERCULOSIS IN CATTLE AND BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2020-09) SANTOSH SAJJAN; SRINIVASA RAO, T (MAJOR); MADHAVAPRASAD, C.B.; RAMANI PUSHPA, R.N.; ASWANI KUMAR, KBrucellosis and tuberculosis are neglected zoonoses having worldwide public health concern. The present study was undertaken to elucidate the prevalence of brucellosis and tuberculosis in cattle and buffaloes, detect and identify the species by PCR, understand the genetic diversity among the detected species, investigate the antimicrobial resistance of pathogens and evaluate the risk factors associated with the occurrence of these diseases. A total of 1070 samples comprising of 330 blood samples, 185 milk samples, 310 nasal swabs and 245 vaginal swabs from 300 cattle and 30 buffaloes which were reared along with cows were collected and examined for detection of brucellosis and mycobacteriosis in different production systems viz., intensive (110), semi-intensive (110) and extensive system (110). The overall prevalence of brucellosis and mycobacteriosis was 6.06% (20/330) and 3.33% (11/330), respectively among the animals studied. The prevalence of both the diseases was higher in farms with intensive production system and larger herd size. Sexually active (2-10 years) and xii aged animals (5-10 years) were at risk of infection for brucellosis and tuberculosis, respectively. Exotic breeds of cattle were more susceptible to mycobacteriosis compared to indigenous breeds. Rose bengal plate test (BPT) and indirect enzyme linked immunosorbent assay (iELISA) tests revealed almost perfect agreement (К=0.92) with each other. While, SIT and IFN-γ ELISA tests revealed substantial agreement (К=0.50) with each other. The species of Brucella recognized by Bruce-ladder multiplex PCR in cattle were found to be B. melitensis (4) and B. abortus (5) out of 09 samples confirmed by PCR. Further, all the Mycobacterium spp. detected by PCR were differentiated as non tuberculous Mycobacterium (NTM) by commercial qPCR kit. Brucella species displayed genetic homogeneity by both PCR-Restriction Fragment Length Polymorphism (RFLP) and Repetitive Element Palindromic (REP)-PCR techniques. Multiple Antimicrobial Resistance (MAR) index for B.abortus and B. melitensis isolates were found to be in the range of 0.66 to 0.83 and 0.5 to 0.91, respectively. Majority of the isolates showed the presence of multiple genes responsible for resistance to rifampicin (rpoB-M4, M5, M6 and +354rB/-720rB gene) fluoroquinalones (gyrA and gyrB). While only one isolate showed the presence of single gene (tetB) responsible for resistance to tetracyclines and one isolate showed presence of single gene {Aac(3)-Ia} responsible for resistance to aminoglycosides. None of the isolates showed presence of catB gene responsible for resistance to chloramphenicol even though all the isolates were resistant to chloramphenicol phenotypically. There was significant association with the individual level (age, sex and breed) and herd level risk factors (production system, herd size, cleanliness and lack of screening of animals) with the occurrence of brucellosis and mycobacteriosis in cattle and buffaloes as evidenced by Odd’s ratio.ThesisItem Open Access STUDIES ON EMERGING ZOONOTIC BACTERIAL PATHOGENS OF FISH AND SHELLFISH FROM FRESH WATER, MARINE AND ESTUARINE SOURCES OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2020-08) SUBHASHINI, NELAPATI; SRINIVASA RAO, T (MAJOR); MADHAVA RAO, T; RAMANI PUSHPA, R.N.; ASWANI KUMAR, KHuman contact with and consumption of fishes presents hazards from a range of bacterial zoonotic infections. The present study was undertaken to characterize Arcobactaer spp., Aeromonas spp. and V. vulnificus of fresh water, estuarine/ brackish and marine origin based on cultural isolation. A total of 420 samples comprising fish and prawn samples from fresh (70 each), marine (70 each) and estuarine environments (70 each) were analyzed to characterize Arcobacter spp., Aeromonas spp. and V. vulnificus and further these samples were screened for antibiotic residues by HPLC. Overall prevalence of Arcobacter spp. was found to be 12.61% (53/420). Out of the 53 Arcobacter spp. isolates, m-PCR revealed 38 (56.71%) to be A. butzleri, 3 (4.48%) to be A. cryaerophilus and 12 (17.91%) to be A. skirrowii. Out of the 53 Arcobacter spp. isolates, virulence genes mviN, irgA, hecA, cj1349, tlyA, pldA, hecB, ciaB and cadF were detected in 75.47%, 9.43%, 3.77%, 30.18%, 98.11%, 92.45%, 11.32%, 94.33% and 81.13% of Arcobacter spp. isolates, respectively. Antibiogram profile of 53 Arcobacter xix spp. isolates revealed natural resistance towards penicillin-G (100%); resistance to vancomycin (75.47%), nalidixic acid (26.42%), erythromycin (18.87%), cefixime and kanamycin (5%) and co-trimoxazole (3.77%). ESBL production was confirmed in 28 Arcobacter spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all the 28 isolates. A greater degree of molecular heterogeneity was observed among ESBL positive Arcobacter butzleri (16) and A. skirrowii (12) isolates, respectively by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. Overall prevalence of Aeromonas spp. was found to be 26.42% (111/420). Out of 111 Aeromonas spp. isolates, m-PCR revealed 98 (88.28%) to be A. veroni, 9 (8.10%) to be A. hydrophila, 2 (1.80%) A. media and 2 (1.80%) A. caviae. Out of 111 Aeromonas spp. isolates, virulence genes act, ast, alt, ahyB, fla, lip, aer, ser, gcat and exu were detected in 53.15%, 4.5%, 5.4%, 9.9%, 0.9%, 22.52%, 16.21%, 2.7% 5.4 % and 15.31% of isolates, respectively. Antibiogram profile of 111 Aeromonas spp. isolates revealed natural resistance towards penicillin-G (100%) and resistance to vancomycin (63.06%) and nalidixic acid (50.45%). ESBL production was confirmed in 12 Aeromonas spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all 12 isolates. A greater degree of molecular heterogeneity was observed among 12 ESBL positive Aeromonas spp. isolates from different sources as 12 different genotypes were observed by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. xx Overall prevalence of V. vulnificus was found to be 6.19% (26/420). All the V. vulnificus isolates carried vvhA gene and none of the isolates were belonging to Bt2 or serovar E. All the V. vulnificus isolates belonged to Bt1. Antibiogram profile of 26 V. vulnificus isolates revealed natural resistance for penicillin-G (100%) and resistance to vancomycin (61.54%), erythromycin (46.15%), cefixime (46.15%) and nalidixic acid (30.77%). ESBL production was confirmed in 17 V. vulnificus isolates by both phenotypic and molecular methods and blaTEM gene was the predominant gene in 16 isolates and blaSHV gene was detected in only one V. vulnificus isolate. A greater degree of molecular heterogeneity was observed among 17 ESBL positive V. vulnificus isolates from different sources as 16 and 17 different genotypes were observed under ERIC-PCR and REP-PCR, respectively. The discriminatory power of the two typing methods for V. vulnificus isolates was found to be highly significant (>0.90) i.e. 0.9926 and one for ERIC and REP-PCR, respectively. Cluster analysis revealed a greater degree of homogeneity and heterogeneity among different isolates (Arcobacter spp., Aeromonas spp. and V. vulnificus) recovered from various sources and indicating that there is a chance of cross-contamination particularly in the fish markets. All the fish and shellfish samples were negative for antibiotic residues by HPLC.ThesisItem Open Access STUDIES ON MOLECULAR EPIDEMIOLOGY OF BRUCELLOSIS AND TUBERCULOSIS IN CATTLE AND BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2020-09) SANTOSH SAJJAN; SRINIVASA RAO, T(MAJOR); MADHAVAPRASAD, C.B.; RAMANI PUSHPA, R.N.; ASWANI KUMAR, KBrucellosis and tuberculosis are neglected zoonoses having worldwide public health concern. The present study was undertaken to elucidate the prevalence of brucellosis and tuberculosis in cattle and buffaloes, detect and identify the species by PCR, understand the genetic diversity among the detected species, investigate the antimicrobial resistance of pathogens and evaluate the risk factors associated with the occurrence of these diseases. A total of 1070 samples comprising of 330 blood samples, 185 milk samples, 310 nasal swabs and 245 vaginal swabs from 300 cattle and 30 buffaloes which were reared along with cows were collected and examined for detection of brucellosis and mycobacteriosis in different production systems viz., intensive (110), semi-intensive (110) and extensive system (110). The overall prevalence of brucellosis and mycobacteriosis was 6.06% (20/330) and 3.33% (11/330), respectively among the animals studied. The prevalence of both the diseases was higher in farms with intensive production system and larger herd size. Sexually active (2-10 years) and aged animals (5-10 years) were at risk of infection for brucellosis and tuberculosis, respectively. Exotic breeds of cattle were more susceptible to mycobacteriosis compared to indigenous breeds. Rose bengal plate test (BPT) and indirect enzyme linked immunosorbent assay (iELISA) tests revealed almost perfect agreement (К=0.92) with each other. While, SIT and IFN-γ ELISA tests revealed substantial agreement (К=0.50) with each other. The species of Brucella recognized by Bruce-ladder multiplex PCR in cattle were found to be B. melitensis (4) and B. abortus (5) out of 09 samples confirmed by PCR. Further, all the Mycobacterium spp. detected by PCR were differentiated as non tuberculous Mycobacterium (NTM) by commercial qPCR kit. Brucella species displayed genetic homogeneity by both PCR-Restriction Fragment Length Polymorphism (RFLP) and Repetitive Element Palindromic (REP)-PCR techniques. Multiple Antimicrobial Resistance (MAR) index for B.abortus and B. melitensis isolates were found to be in the range of 0.66 to 0.83 and 0.5 to 0.91, respectively. Majority of the isolates showed the presence of multiple genes responsible for resistance to rifampicin (rpoB-M4, M5, M6 and +354rB/-720rB gene) fluoroquinalones (gyrA and gyrB). While only one isolate showed the presence of single gene (tetB) responsible for resistance to tetracyclines and one isolate showed presence of single gene {Aac(3)-Ia} responsible for resistance to aminoglycosides. None of the isolates showed presence of catB gene responsible for resistance to chloramphenicol even though all the isolates were resistant to chloramphenicol phenotypically. There was significant association with the individual level (age, sex and breed) and herd level risk factors (production system, herd size, cleanliness and lack of screening of animals) with the occurrence of brucellosis and mycobacteriosis in cattle and buffaloes as evidenced by Odd’s ratio.ThesisItem Open Access STUDIES ON EMERGING ZOONOTIC BACTERIAL PATHOGENS OF FISH AND SHELLFISH FROM FRESH WATER, MARINE AND ESTUARINE SOURCES OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2020-08) SUBHASHINI, NELAPATI; SRINIVASA RAO, T(MAJOR); MADHAVA RAO, T; RAMANI PUSHPA, R.N.; ASWANI KUMAR, KHuman contact with and consumption of fishes presents hazards from a range of bacterial zoonotic infections. The present study was undertaken to characterize Arcobactaer spp., Aeromonas spp. and V. vulnificus of fresh water, estuarine/ brackish and marine origin based on cultural isolation. A total of 420 samples comprising fish and prawn samples from fresh (70 each), marine (70 each) and estuarine environments (70 each) were analyzed to characterize Arcobacter spp., Aeromonas spp. and V. vulnificus and further these samples were screened for antibiotic residues by HPLC. Overall prevalence of Arcobacter spp. was found to be 12.61% (53/420). Out of the 53 Arcobacter spp. isolates, m-PCR revealed 38 (56.71%) to be A. butzleri, 3 (4.48%) to be A. cryaerophilus and 12 (17.91%) to be A. skirrowii. Out of the 53 Arcobacter spp. isolates, virulence genes mviN, irgA, hecA, cj1349, tlyA, pldA, hecB, ciaB and cadF were detected in 75.47%, 9.43%, 3.77%, 30.18%, 98.11%, 92.45%, 11.32%, 94.33% and 81.13% of Arcobacter spp. isolates, respectively. Antibiogram profile of 53 Arcobacter spp. isolates revealed natural resistance towards penicillin-G (100%); resistance to vancomycin (75.47%), nalidixic acid (26.42%), erythromycin (18.87%), cefixime and kanamycin (5%) and co-trimoxazole (3.77%). ESBL production was confirmed in 28 Arcobacter spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all the 28 isolates. A greater degree of molecular heterogeneity was observed among ESBL positive Arcobacter butzleri (16) and A. skirrowii (12) isolates, respectively by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. Overall prevalence of Aeromonas spp. was found to be 26.42% (111/420). Out of 111 Aeromonas spp. isolates, m-PCR revealed 98 (88.28%) to be A. veroni, 9 (8.10%) to be A. hydrophila, 2 (1.80%) A. media and 2 (1.80%) A. caviae. Out of 111 Aeromonas spp. isolates, virulence genes act, ast, alt, ahyB, fla, lip, aer, ser, gcat and exu were detected in 53.15%, 4.5%, 5.4%, 9.9%, 0.9%, 22.52%, 16.21%, 2.7% 5.4 % and 15.31% of isolates, respectively. Antibiogram profile of 111 Aeromonas spp. isolates revealed natural resistance towards penicillin-G (100%) and resistance to vancomycin (63.06%) and nalidixic acid (50.45%). ESBL production was confirmed in 12 Aeromonas spp. isolates by both phenotypic and molecular methods and blaTEM was the only β-lactamase gene detected in all 12 isolates. A greater degree of molecular heterogeneity was observed among 12 ESBL positive Aeromonas spp. isolates from different sources as 12 different genotypes were observed by ERIC-PCR and REP-PCR. The discriminatory power of the two typing methods for Arcobacter spp. was found to be highly significant (>0.90) i.e. one. Overall prevalence of V. vulnificus was found to be 6.19% (26/420). All the V. vulnificus isolates carried vvhA gene and none of the isolates were belonging to Bt2 or serovar E. All the V. vulnificus isolates belonged to Bt1. Antibiogram profile of 26 V. vulnificus isolates revealed natural resistance for penicillin-G (100%) and resistance to vancomycin (61.54%), erythromycin (46.15%), cefixime (46.15%) and nalidixic acid (30.77%). ESBL production was confirmed in 17 V. vulnificus isolates by both phenotypic and molecular methods and blaTEM gene was the predominant gene in 16 isolates and blaSHV gene was detected in only one V. vulnificus isolate. A greater degree of molecular heterogeneity was observed among 17 ESBL positive V. vulnificus isolates from different sources as 16 and 17 different genotypes were observed under ERIC-PCR and REP-PCR, respectively. The discriminatory power of the two typing methods for V. vulnificus isolates was found to be highly significant (>0.90) i.e. 0.9926 and one for ERIC and REP-PCR, respectively. Cluster analysis revealed a greater degree of homogeneity and heterogeneity among different isolates (Arcobacter spp., Aeromonas spp. and V. vulnificus) recovered from various sources and indicating that there is a chance of cross-contamination particularly in the fish markets. All the fish and shellfish samples were negative for antibiotic residues by HPLC.ThesisItem Open Access Characterization of MDR Enterococcus species of animal, human and environmental origin with special emphasis on vancomycin resistance(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) CHAITANYA, GOTTAPU; BINDU KIRANMAYI, CH (MAJOR); SRINIVASA RAO, T; ASWANI KUMAR, KThe present study was undertaken to characterize Enterococcus species of animal, human and environmental origin based on cultural isolation. It was also aimed to determine the major antibiotic resistant genotypes (vancomycin resistance, high level aminoglycoside resistance and β- lactamase resistance), phenotypic virulence factors and virulence genes of Enterococcus species of worldwide public health importance. A total of 780 samples were collected including animals, foods of animal origin, water, human faecal, human diarrhoeic and human urine samples and were examined for presence of Enterococcus spp. i.e. E. faecalis, E. faecium, E. gallinarum and E. casseliflavus. Overall prevalence of genus Enterococcus was found to be 86.79% and the prevalence of the Enterococcus spp. in various samples ranging from 100% each in sheep rectal swabs, pig rectal swabs and human diarrhoeic samples to 55.00% in uterine discharges of cattle. Enterococci are opportunistic pathogens and cause occasional infections. Virulence gene and phenotypic virulence factors are major indicators to pathogenicity of these microorganisms. In present study, presence of phenotypic virulence factors among 608 Enterococcus isolates i.e. hemolysis of sheep RBC, slime layer formation, lipase activity, caseinase activity, biofilm formation, gelatinase, DNase activity and HA test were detected in 312 (51.31%), 243 (39.96%), 47 (7.73%), 121 (19.90%), 236 (38.81%), 141 (23.19%), 37 (6.08%) and 87 (14.30%) of Enterococcus isolates, respectively. Out of seven virulence markers investigated in Enterococcus isolates, gelE was predominant in 181 isolates (29.76%) followed by 180 asa (29.60% each), 131 hyl (21.54%), 117 ace (19.24%), 101 efaA (16.61%) and 68 cyl (11.18%). Antibiogram profiling of 608 isolates revealed a major fraction of the Enterococcus isolates to be resistant to polymixin-B (95.55%) followed by ceftazidime (93.25%), erythromycin (77.63%) and streptomycin (44.07%). A total of 179 (29.44%) isolates were positive for HLAR genes and aac(6´)Ie-aph(2˝)Ia was the only gene detected in all isolates and 127 (20.88%) isolates were positive for blaZ gene. The blaZ gene was predominantly detected in E. faecium (34.63%), followed by E. faecalis (14.38%), E. gallinarum (16.50%) and E. casseliflavus (16.66%). A total of 608 Enterococcus isolates studied, 125 Enterococcus isolates were identified as VRE genotypically. The genes VanB, VanC1 and VanC2 were detected in 14 (11.20%), 69 (55.20%) and 42 (36.60%) Enterococcus isolates, respectively. None of isolates showed VanA gene. A greater degree of heterogeneity was observed among 124 VRE isolates (one E. gallinarum isolate did not yield any bands for both ERIC-PCR and REP- PCR) of different species from different sources as revealed by presence of 122 genotypes and 123 genotypes by ERIC and REP-PCR analysis, respectively. Nineteen different E. faecalis, 15 E. faecium, 57 E. gallinarum and 31 E. casseliflavus subtypes were differentiated by ERIC-PCR, whereas 21 different E. faecalis, 15 E. faecium, 56 E. gallinarum and 31 E. casseliflavus subtypes by REP- PCR. Genotyping of VR Enterococcus species by ERIC- PCR and REP- PCR was found to be highly significant since discriminatory power >0.9 are considered highly significant (0.9997 for ERICPCR and 0.9999 for REP-PCR. Cluster analysis also revealed a great degree of homogeneity among some VRE isolates recovered from different sources and implied at the chance of cross-contamination of foods of animal originThesisItem Open Access STUDIES ON SALMONELLA SEROVARS OF ANIMAL ORIGIN WITH SPECIAL REFERENCE TO FOOD BORNE SALMONELLA SEROVARS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) SURESH, YASARLA; BINDU KIRANMAYI, Ch.(MAJOR); SRINIVASA RAO, T; Srivani, MSalmonella is a foodborne pathogen having a worldwide public health concern. The present study was undertaken to characterize Salmonella species of animal origin based on cultural isolation, molecular confirmation of serovars, their virulence profile and antibiogram using PCR and genetic diversity studies by employing ERIC-PCR and REP-PCR. A total of 516 samples comprising poultry cloacal swabs (249), raw foods of animal origin (118 chicken samples, 65 mutton and 30 pork), 17 poultry liver swabs) and 37 poultry farm water samples were examined for presence of Salmonella serovars. Overall prevalence of Salmonella isolates was found to be 4.06% (21/516) with highest prevalence in chicken samples (6/118, 5.08%) followed by cloacal swabs of poultry (12/249, 4.81%), mutton (2 /65, 3.07%) and pork (1/30, 3.33%). All the isolates carried all the 7 virulence genes i.e. invA, invH, sopB, sopE & stn (21/21, 100%), while pefA genes was found only in S. Typhimurium isolates and sefC gene was found only in S. Enteritidis isolates (2). Antibiogram of Salmonella isolates revealed 100% susceptibility to co- trimoxazole and polymyxin–B, intermediate resistant against ampicillin (28.57%), cefotaxime (19.04%), gentamycin (14.28%), amikacin (9.52%), ceftriaxone (9.52%), ciprofloxacin (9.52%), tetracycline (4.76%) and streptomycin (4.76%) while higher resistance was observed towards amikacin (61.90%) followed by ampicillin (52.30%), tetracycline (38.09%), ceftriaxone (33.33%), gentamicin, sulfamethoxazole,cefpotaxime and nalidixic acid (28.57% each), ciprofloxacin (23.80%), doxycycline hydrochloride and chloramphenicol (19.04% each) and streptomycin (9.52%). Of the 21 Salmonella isolates, 15 isolates were found resistant to β-lactam antibiotics like ceftriaxone (33.33%), cefotaxime (28.57%), aztreonam (23.80%) and ceftazidime (23.80%) was detected. β- lactamase genes were detected in a total of 11 isolates (11/21, 52.38%), blaTEM being the predominant gene detected (9/11, 81.18%), followed by blaCTX-M group II (2/11, 18.18%), blaOXA (1/11, 9.09%) and blaCTX-M group IX (1/11, 9.09%) and no single isolate showed blaCTX-M group 1 and blaSHV genes. ERIC PCR and REP-PCR analysis revealed a greater degree of heterogeneity among S.Typhimurium and Salmonella group II isolates from different sources. ERIC PCR genotyping distinguished 7 isolates each of S.Typhimurium and Salmonella group II into 6 genotypes each whereas REP-PCR distinguished all the isolates into distinct genotypes. The discriminatory power of ERIC-PCR and REP-PCR for Salmonella isolates was found to be highly significant (>0.9) i.e. 0.952 and 1.0, respectively.ThesisItem Open Access DETECTION OF PROTEUS MIRABILIS IN FOODS OF ANIMAL ORIGIN, ANIMAL AND HUMAN CLINICAL SAMPLES AND WATER WITH SPECIAL EMPHASIS ON β-LACTAMASES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) PRASASTHA RAM, V; VENKATESWARA RAO, L(MAJOR); SRINIVASA RAO, T; SUBRAMANYAM, K.V.P. mirabilis is an emerging foodborne pathogen having worldwide public health concern. The present study was undertaken to characterize P. mirabilis species of animal and human origin based on cultural isolation, PCR detection, antibiogram, virulence profiles and genetic diversity. A total of 507 samples comprising foods of animal origin (215), faecal swab samples (188), human urine samples (65), human diarrhoeic stool samples (12) as well as water samples (27) were examined. Overall prevalence of P. mirabilis was found to be 34.51% (175/507) by species-specific PCR. Among foods of animal origin, the highest rate of P. mirabilis isolates were recovered from chicken samples (38.7%), followed by pork (37.5%) and mutton samples (28.9%). Among faecal swabs of livestock, the highest rate of P. mirabilis isolates were recovered from poultry (49%), followed by pigs (37.8%). Human urine samples showed a prevalence rate of 10.7%. Water samples showed 7.4% prevalence. All the human diarrhoeic stool samples were negative for P. mirabilis. All the P. mirabilis isolates carried a combination of putative virulence genes. The genes ureC, ureA, flaA, hpmA and zapA were detected in 80.5%, 72.5%, 28.5%, 60.5% and 50.28% of P. mirabilis isolates, respectively. Antibiogram of P. mirabilis isolates revealed sensitivity towards gentamicin (76.57%), followed by ampicillin (64.57%), kanamycin (61.14%), amikacin (60.57%) and streptomycin (43.42%). Higher resistance was observed for erythromycin (71.42%), nalidixic acid (62.85%), ciprofloxacin (62.85%), tetracycline (60%), polymyxin-B (60%), cefoxitin (49.14%) and amikacin (36%). Notable percentages of isolates were intermediately resistant against streptomycin (33.14%), erythromycin (20.57%) and cefoxitin (18.28%). β-lactamase genes were detected in a total of 23 isolates (13.14%). Prevalence rates of β-lactamase genes among different samples was 23.6%, 11.1%, 10.8% and 42.8% from chicken, pork, poultry cloacal swabs and human urine samples, respectively with blaTEM being the predominant gene detected (69.56%) followed by blaOXA (26.08%), blaAmpC gene FOX (13.04%), blaCTX-M group I (4.34%), blaSHV (4.34%) and blaAmpC gene CIT (4.34%) among all the tested P. mirabilis isolates. Of the twenty-three P. mirabilis isolates analyzed, twenty-three ERIC-PCR patterns and twenty-two REP-PCR patterns were obtained. A pair of P. mirabilis isolates (13 and 14) that had identical REP-PCR pattern (R13) were distinguished by ERIC-PCR into two different genotypes (E13 and E14). The two P. mirabilis isolates sharing identical REP-PCR pattern (R13) but differential ERIC-PCR pattern (E13 and E14) were recovered from poultry cloacal swabs (PC 4 and PC 5) collected from LFC, Gannavaram. The discriminatory power ERIC-PCR and REP-PCR for P. mirabilis isolates was found to be 1 and 0.996, respectively. Close clustering between P. mirabilis of animal and human origin are indicative of probable zoonotic significance.ThesisItem Open Access CHARACTERIZATION OF Campylobacter SPECIES OF ANIMAL AND HUMAN ORIGIN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) SRINIVAS, K; SRINIVASA RAO, T(MAJOR); BINDU KIRANMAYI, Ch.; LAKSHMI KAVITHA, KCampylobacter is a well established food-borne zoonotic pathogen having a worldwide public health importance. The present study was undertaken to characterize Campylobacter species of animal and human origin based on cultural isolation. A total of 513 samples comprising faecal samples of livestock and poultry (174), intestinal contents of livestock and poultry (96), foods of animal origin (185) and human diarrhoeic samples (35) as well as samples of miscellaneous origin (23) were examined. Overall prevalence of Campylobacter spp. was found to be 4.87% (25/513) with highest prevalence in poultry intestinal samples (8/36, 22.2%), followed by pig intestinal contents (4/40, 10%), pig faecal samples (5/60, 8.33%), pork samples (3/90, 3.33%), human diarrhoeic samples (1/35, 2.85%), poultry faecal samples (3/114, 2.63%) and chicken meat samples (1/50, 2%). All 7 virulence genes under the study (iam, virB11, flaA, cadF, cdtA, cdtB, cdtC) were detectable. flaA and cadF genes were present in all the isolates (25/25, 100%) followed by iam (23/25, 92%), cdtA (20/25, 80%), cdtB (20/25, 80%), cdtC (17/25, 68%) and virB11 (4/25, 16%). Antibiogram profiling of 25 isolates revealed that a major fraction of the Campylobacter isolates showed sensitivity to colistin and co-trimoxazole (64%), chloramphenicol and azithromycin (56%) and ceftazidime (52%). All the Campylobacter isolates were resistant to at least one of the antibiotics tested. Higher resistance was observed for vancomycin and tetracycline (76%), cephalothin and ciprofloxacin (68%), erythromycin (56%) and doxycycline (52%). Notable percentages of isolates were intermediately resistant against nalidixic acid and nitrofurantoin (44%), gentamicin and streptomycin (32%) and clindamycin (28%). Resistance to β-lactam antibiotics like aztreonam (40%), cefotaxime (32%), ceftriaxone (40%) and ceftazidime (28%) were detected. ESBL production was confirmed phenotypically in 12 isolates by CDM . blaTEM was the only β-lactamase gene detected in 60% of the isolates. rep-PCR (ERIC-PCR and REP-PCR) and flaA-PCR-RFLP analysis revealed a greater degree of molecular heterogeneity among Campylobacter isolates from different sources. ERIC-PCR genotyping revealed 6 and 18 genotypes distinguished among 6 and 19 isolates of C. jejuni and C. coli, respectively. REP-PCR genotyping revealed 5 and 19 genotypes among 6 and 19 isolates of C. jejuni and C. coli., respectively while flaA PCR-RFLP typing of 6 isolates of C. jejuni and 19 C. coli isolates revealed 5 and 15 genotypes, respectively. The discriminatory power of the three typing methods for Campylobacter species were found to be highly significant (>0.90) i.e. 0.9967 (ERIC PCR and REP-PCR) and 0.9833 (flaA-PCR-RFLP). Cluster analysis revealed a great deal of homogeneity and heterogeneity among different isolates recovered from various sources and hinted at the chance of cross-contamination of foods of animal origin.ThesisItem Open Access DETECTION OF EXTENDED SPECTRUM βLACTAMASES IN SHIGA TOXIN PRODUCING ESCHERICHIA COLI ISOLATED FROM DIFFERENT RAW MEAT SAMPLES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-10) PRABHU KISHORE, G; SRINIVASA RAO, T(MAJOR); BINDU KIRANMAYI, Ch.; RAMANI PUSHPA, R.N.Shiga toxin producing Escherichia coli is an important foodborne pathogen having worldwide public health concern. The present study was undertaken to characterize STEC from raw meats of animal origin and human origin based on cultural and biochemical isolation, PCR detection, virulence profiles, antibiogram, serotyping and genetic diversity. A total of 523 samples comprising raw foods of animal origin (470) and human diarrhoeic stools (53) were examined. Out of 523 total samples collected, 178 (34.03%) samples were positive for Escherichia coli by cultural and molecular methods. Out of 178 E. coli isolates, 154/470 (32.76%) isolates were from raw foods of animal origin and 24/53 (45.28%) from human diarrhoeic stool samples.Out of 470 food samples screened,the highest rate of E. coli isolation was recorded from mutton (47.27%, 26/55), followed by cara beef (33.33%, 13/39) and chicken (30.58%, 115/376). Out of a total of 178 E. coli isolates, m-PCR revealed 43 (24.1%) isolates as STEC. Among the 43 STEC isolates, 9 (34.6%) were from mutton samples, 6 (46.1%) cara beef samples, 19 (16.5%) chicken samples and 9 (37.5%) human isolates. The genes stx1, stx2, eaeA and hlyAwere detected in 93.02%, 39.53%, 44.18% and 46.51% of STEC isolates, respectively. Two mutton samples showed all the four targeted genes in present study. Antibiogram study of STEC isolates revealed sensitivity towards fosfomycin (100%) chloramphenicol (95.3%), gentamicin (55.8%) and Amikacin (53.4%). Higher resistance was observed for vancomycin (100%), erythromycin (100%), streptomycin (100%), ciprofloxacin (95.3%), naladixic acid (93%), Ampicillin (90.6%), cefotaxime (86%), ceftazidime (81.3%), ceftriaxone (74.1%) and co-trimoxazole (58.1%). Notable percentages of isolates were intermediately resistant against norfloxacin (32.5%), kanamycin and tetracycline (27.9% each)and aztreonam (16.2%). ESBL phenotypes were confirmed in a total of 21 STEC isolates using phenotypic confirmation tests. β-lactamase genes were detected in a total of 21(48.8%) STEC isolates, with blaTEM being the predominant gene detected (25.5%, 11/43) followed by blaCTX-M group I (20.9%, 9/43), blaOXA(13.9%, 6/43), blaSHV(9.3%, 4/43), blaCTX-M group II ( 4.6%, 2/43) and no single isolate showed blaAmpCgene among all the tested STEC isolates. Prevalence rates of βlactamase genes among different samples is 57.8%, 44.4%, 50% and 33.3% among the STEC isolates obtained from raw meat of chicken, mutton, cara beef and human diarrhoeic stool samples, respectively. CTX-M beta-lactamase was found to be the most frequent mechanism of ESBL resistance in STEC isolates of the present study. All the 21 ESBL producing STEC isolates were sent for serotyping on the basis of ‘O’ antigen and 9 of them were belonging to serotype O22, 4 were serotype O88 and others were ONT. ERIC PCR and REP-PCR analysis revealed a greater degree of heterogeneity among STEC isolates from different and within the same sources. Of the 21 ESBL producing STECisolates analyzed, 21 ERIC-PCR patterns and 21 REP-PCR patterns were obtained by using ERIC and REP-PCR analysis, respectively. The discriminatory power for both ERIC and REP-PCR was found to be 0.999. Genetic diversity of STEC recovered from foods of animal origin and humans in India adds to the heterogeneity reports among STECspecies world-wide, supporting diversity among same species. Close clustering between STECfrom raw meats of animal origin and human origin is apossible indicative of probable cross contamination and its zoonotic significance.
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