OCCURRENCE AND CHARACTERIZATION OF ENTEROCOCCUS SPECIES ISOLATED FROM LIVESTOCK AND VEGETABLE FARMS IN ZARIA, NIGERIA

OCCURRENCE AND CHARACTERIZATION OF ENTEROCOCCUS SPECIES ISOLATED FROM LIVESTOCK AND VEGETABLE FARMS IN ZARIA, NIGERIA

Abstract

This study was carried out to determine the occurrence of Enterococcus species and characterize Enterococcus faecalis and E. faecium isolates from poultry, cattle and vegetable farms in Zaria, Nigeria. A total of 670 samples consisting of cattle rectal swabs and manure (n=130 and 65 respectively), poultry cloacal swabs and manure (n=130 and 65 respectively), irrigation water (n=40), vegetables (52 lettuce, 20 cabbage and 48 carrot) and soil (n=120). The samples were collected between January, and July, 2014 in Zaria, Nigeria. Isolation and speciation was carried out using standard microbiological techniques and further confirmation of E. faecium isolates using polymerase chain reaction (PCR). Further characterization of the isolates identified as E. faecium and E. faecalis was carried out by detection of phenotypic expression of virulence and resistance traits, detection of some of the corresponding genes by PCR and transferability of some resistance traits. Sequencing of the pbp5 gene was also carried out to determine the amino acid substitutions responsible for high level ampicillin resistance among the isolates. Enterococcus was isolated from 200 (29.9%) out of the 670 samples collected. Two hundred and sixty seven enterococcal isolates were recovered from the 200 samples. Except for irrigation water, Enterococcus was isolated from all sample types. Nine species; E. faecium (n=130), E. faecalis (n=5), E. raffinosus (n=6), E. avium (n=1), E. gallinarum (n=48), E. casseliflavus (n=3), E. mundtii (n=4), E. durans (n=1) and E. hirae (n=5) were differentiated from the 267 enterococcal isolates, while 64 were unidentifiable. The isolation rates were Enterococcus faecium (48.7%) followed by E. gallinarum (17.9%) and were the predominant species isolated from all sample types (except for lettuce). Virulence factors observed were haemolysin (20.7%), gelatinase (9.6%), aggregation substance (1.48%) and biofilm (83.7%) which were expressed in addition to carriage of their corresponding genes; cylA (1/19), gelE (10/19) and asa1 (14/20) except for enterococcal suface protein (esp) gene. However, a silent gelE gene was detected in 2 isolates. High percentages of resistance were detected for erythromycin (54.1%), tetracycline (52.6%) and ampicillin (46.7%) and lower values were observed for quinupristin/dalfoprsitin (3.70%), chloramphenicol (6.7%) and ciprofloxacin (8.1%). All the isolates were susceptible to vancomycin. MIC of gentamicin and ampicillin above resistance breakpoints (≥500μg/ml and ≥16μgml, respectively) were observed among the high level gentamicin (35/37) and ampicillin (7/63) resistant isolates. Multiple drug resistance (MDR) to three to six different antibiotics was observed (45.9%) among the isolates tested, with majority of the MDR originating from poultry sources. Twenty four different MDR patterns were identified with the CN-E-S-TE phenotype (11 isolates) followed by AM-CN-E-S-TE (seven isolates) being the predominant MDR pattern. Resistance to AM, E and TE frequently occurred among the 24 different MDR phenotypes. Genes responsible for resistance to gentamicin: aac(6′)-Ie-aph(2”) (19/19), aph(2′)-Ic (8/9), aph(3′)-IIIa (9/10) and ant(4′) (4/10), ampicillin; pbp5, erythromycin; erm(B) (19/19) and tetracycline: tet(K) (9/10) tet(L) (20/20) tet(M) (18/19) and tet(O) (10/10) were detected among the E. faecium and E. faecalis isolates tested. In addition to co-carriage of more than one gene (aminoglycoside modifying genes and tetracycline resistant genes) mediating resistance to the same antibiotic, co-carriage of the erm(B)+tet gene in a single strain was observed among most of the isolates. Five of the seven E. faecium isolates presented a total of nine amino acid substitutions in PBP5 protein. Four new amino acid substitution; 500Glu→Leu, 502Asp→Arg, 612IIe→X and 614IIe→Phe in addition to known polymorphism 470His→Gln, 496Asn→Lys, 497Phe→IIe, 499Ala→Thr, 525Glu→Asp resulted from mutations of the pbp5 gene. In vitro intra and inter species transfer of determinants coding for either or both of erythromycin and tetracycline resistance to recipient cells was observed in six (75%) out of the eight isolates tested. In conclusion, enterococci from livestock and vegetable farms in Zaria harbour a variety of virulence and antimicrobial resistance determinants that give them the potential to cause antimicrobial resistant nosocomial infections. They may also serve as reservoirs of virulence and antimicrobial resistance genes of bacteria that could eventually be transmitted either to other bacteria, animals or even to humans

OCCURRENCE AND CHARACTERIZATION OF ENTEROCOCCUS SPECIES ISOLATED FROM LIVESTOCK AND VEGETABLE FARMS IN ZARIA, NIGERIA

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