Potential of a polyherbal drug to prevent antimicrobial resistance in bacteria to antibiotics.

Persistence of antibacterial drugs for prolonged period in milk increases the probability of antimicrobial resistance progress. Ceftizoxime was found to be excreted in milk for a prolonged period in goats, cows and buffaloes following intravenous injection of ceftriaxone and ceftizoxime. A single dose of ceftriaxone was administered intravenously in healthy control goats (group I) and a single oral dose of the commercial mammary protective polyherbal drug (1.9 gm) was given one hour prior to intravenous ceftriaxone injection in healthy (group II) and induced mastitic (group III) goats to evaluate milk disposition of ceftizoxime following single intravenous dosing of ceftriaxone at 42.25 mg kg-1.Ceftriaxone/ceftizoxime was analyzed by HPLC. The t1/2α and t1/2ß values were 14.755 ± 2.733 and 149.079 ± 18.565 hour, respectively indicating prolonged persistence of ceftizoxime in milk. The polyherbal drug increased the milk concentration at later hours and hastened the excretion of ceftizoxime from milk compared to control group. Ceftriaxone could not be detected in milk. The study suggested that adjunct single or repeated therapy of  the polyherbal drug may cause non persistence of ceftriaxone and shorter persistence of ceftizoxime in milk.

Genomic Identity of Fluoroquinolone-Resistant blaCTX-M-15-Type ESBL and pMAmpC ß-Lactamase Producing Klebsiella pneumoniae from Buffalo Milk, India.

We investigated the occurrence of extended-spectrum ß-lactamase (ESBL) and AmpC-type ß-lactamase (ACBL) producing quinolone-resistant Klebsiella pneumoniae (KP) in milk samples of apparently healthy buffaloes (n = 348) and buffaloes (n = 19) with evidence of subclinical mastitis from seven districts of West Bengal, India. In total, 12 ESBL producing KP were isolated with blaCTX-M-15 gene and 7 of them were ACBL producers, as well. The blaCTX-M-15 genes were carried by transposable element ISEcp1. The plasmid-mediated quinolone resistance genes-qnrS, qnrA, qnrB, qepA, and aac(6')-Ib-cr were detected in five, one, three, four, and one isolate (s), respectively. In addition, eight isolates carried mutation in gyrase (gyrA) and six in topoisomerase IV (parC). Resistance markers/genes for sulfonamide (sul1), tetracycline [tet(A) and tet(B)], and aminoglycoside (aacC2) were also detected in eight, four, and one isolate(s), respectively. The class I integrons identified in five isolates carried aad2/aad5 and dfrA12/dfrA17 gene cassettes. The enterobacterial repetitive intergenic consensus-PCR revealed that all the isolates were genetically diverse and comprised a heterogeneous population. Isolation of multidrug-resistant KP, a typical nosocomial pathogen from buffalo milk, reiterates the need to monitor farm animals for ESBL producing Enterobacteriaceae and emphasizes on judicious use of antibiotics in animal husbandry sector.

Prevalence of CTX-M-Producing Klebsiella spp. in Broiler, Kuroiler, and Indigenous Poultry in West Bengal State, India.

This study was undertaken to detect the prevalence of CTX-M-producing Klebsiella spp. in healthy broiler, indigenous, and kuroiler birds reared in West Bengal (India) during November 2014-February 2015. In addition to CTX-M gene, the study was also conducted to reveal the occurrence of other ß-lactamase and class I integron genes in Klebsiella spp. isolates along with their clonal relationship. A total of 321 cloacal swabs from healthy broiler, indigenous, and kuroiler birds were collected from different places of West Bengal, India. Klebsiella spp. isolation rate varies among different types of poultry birds (43.8-72.3%). In total, 33 (10.7%) Klebsiella spp. isolates were detected phenotypically as CTX-M producers and all the isolates possessed blaCTX-M in polymerase chain reaction. Whereas 17 (51.5%) and 16 (48.5%) Klebsiella spp. isolates possessed blaSHV, and blaTEM with blaCTX-M, respectively. None of the CTX-M-producing Klebsiella spp. isolates in this study possessed class I integron gene. Randomly amplified polymorphic DNA-based phylogenetic tree revealed the presence of clonal relationship among the CTX-M-producing Klebsiella spp. isolates, recovered from broilers and indigenous birds. This study identified broilers and indigenous game birds as a potential reservoir of CTX-M-producing Klebsiella spp., which could be transmitted to the human food chain directly or indirectly.

First Report on Vancomycin-Resistant Staphylococcus aureus in Bovine and Caprine Milk.

The present investigation was carried out to study the vancomycin resistance pattern of Staphylococcus aureus isolates (n = 274) obtained from 352 milk samples of bovine (269) and caprine (63) clinical and subclinical mastitis from different districts of West Bengal, India. Of them, seven isolates (vancomycin-resistant S. aureus [VRSA] 1-7) exhibited resistance to vancomycin. Minimum inhibitory concentration of vancomycin (MICvan) for VRSA2 and VRSA3 was ≥16 µg/ml; thus categorized as VRSA. For rest of the isolates, MICvan was 8 µg/ml and they were grouped as vancomycin intermediate S. aureus (VISA). Even though all the isolates were resistant to cefoxitin and oxacillin and possessed mecA gene, none of them carried vancomycin resistance gene. Furthermore, all the seven isolates were subjected to Staphylococcal cassette chromosome mec (SCCmec) typing, Staphylococcal protein A (spa) typing, and enterobacterial repetitive intergenic consensus polymerase chain reaction. All the isolates except VRSA3 and VRSA4 from Kolkata district exhibited diverse genetic lineage, irrespective of their host and antibiotic resistance pattern. These two isolates showed clonal similarity (MRSA-SCCmec-V-spa t267) with methicillin-resistant S. aureus (MRSA) strains previously reported in human and animal infection. Isolation of VRSA and VISA could probably be due to intensive use of vancomycin in healthcare premises, which might have led to the development of glycopeptide-resistant strains and thereafter, further disseminated in the environment, including livestock farms. Detection of VRSA in milk is a serious concern as it may further cause health problems in the consumers. This is the first ever report of VRSA in food animals, even though the pathogen is otherwise prevalent in humans.

Approaches to characterize extended spectrum beta-lactamase/beta-lactamase producing Escherichia coli in healthy organized vis-a-vis backyard farmed pigs in India.

The study was undertaken to investigate the occurrence and to characterize the ESBL/beta-lactamase producing-Escherichia coli in healthy pigs of organized and backyard farms in West Bengal, India. Total 200 rectal swabs were collected randomly from healthy pigs maintained in four organized farms and 10 backyard farms (n=100 each) and 76 isolates were identified as E. coli from organized (48/100, 48%) and backyard pigs (28/100, 28%). Twelve E. coli isolates (6%) in the present study were detected to possess any of the ESBL/beta-lactamase genes studied. ESBL/beta-lactamase producers were isolated with significantly more frequency from backyard pigs than the organized farm pigs (p=0.026). Six of ESBL/beta-lactamase producing isolates were phenotypically confirmed as CTX-M producers and ten of them were confirmed as TEM/SHV producers. PCR and sequencing of the amplified product from representative isolates revealed the presence of blaCTX-M-9, blaSHV-12 and blaTEM-1. No unique combination of the studied beta lactamase genes for organized and backyard farm pig isolates was noted. The ESBL isolates belonged to O13, O55, O133, O153, O157, O158, O166, rough and OUT serogroups. The association of heat labile toxin (elt) (p<0.0005) with organized farm isolates and heat stable toxin (estA) (p=0.0143) with backyard piggery sector was significantly higher. The ESBL/beta-lactamase producers from organized farm (Ak/Ex) and indigenous pigs (Ak/Ex/Te; Ak/CoT/G) showed a characteristic phenotypical antibiotic resistance pattern. Two pairs of isolates from organized and backyard farm pigs showed clonal relationship indicating a possible transmission between the farms which were situated adjacently. Thus the present study revealed backyard farm pigs as major source of ESBL/beta-lactamase producing-E. coli associated with STa and characteristic antibiotic resistance pattern in India.

Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India.

The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks (Poephagus grunniens) with haemorrhagic diarrhoea.

OBJECTIVE: To evaluate the antimicrobial efficacy of berberine, a plant alkaloid. METHODS: Five multi-drug resistant (MDR) STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study. Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods. The binding kinetics of berberine to DNA and protein was also enumerated. RESULTS: For both categories of enterovirulent Escherichia coli (E. coli) isolates, berberine displayed the antibacterial effect in a dose dependent manner. The MIC(50) of berberine chloride for STEC/EPEC isolates varied from 2.07 µM to 3.6 µM with a mean of (2.95 ± 0.33) µM where as for ETEC strains it varied from 1.75 to 1.96 µM with a mean of (1.87 ± 0.03) µM. Berberine bind more tightly with double helix DNA with Bmax and Kd of (24.68±2.62) and (357.8±57.8), respectively. Berberine reacted with protein in comparatively loose manner with Bmax and Kd of (18.9±3.83) and (286.2±113.6), respectively. CONCLUSIONS: The results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E. coli.

Virulence repertoire of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic Escherichia coli (ETEC) from diarrhoeic lambs of Arunachal Pradesh, India.

A total of 107 faecal samples were collected from diarrhoeic lambs of high altitude terrains (2,000 to 5,000 m above the mean sea level) of Tawang and West Kameng districts of Arunachal Pradesh, India. Total 234 Escherichia coli were isolated and further subjected to PCR for the study of virulence repertoire characteristics of Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC). Out of the 234 isolated E. coli, 32% were found positive for STEC, and 9% were carrying virulence gene for ETEC. The isolated STEC serogroups were O159, O127, O120, O113, O60, O30, O25, O8 and O2. Of all the 74 STEC strains, PCR showed that 18% isolates carried stx ( 1 ), 26% possessed stx ( 2 ) and 47% produced positive amplicon for both. Other virulent attributes like intimin (eaeA), enterohaemolysin (ehxA) and STEC auto-agglutinating adhesin (saa) were present in 18%, 43% and 44% of the isolates, respectively. The isolated ETEC serogroups were O172, O170, O159, O146, O127, O120, O113, O86, O75, O60, O30, O25, O8, O2, OR and OUT. Of the 22 ETEC-positive isolates, 23%, 18% and 4.5% possessed the gene only for LT, STa and STb, respectively, whereas 54% carried genes for both LT and STb. Some serogroups of E. coli like O159, O127, O120, O113, O60, O30, O25, O8 and O2 possessed genes for both Shiga toxin and enterotoxin. This study is the first report of ETEC isolation from diarrhoeic lambs in India. The moderately high proportion of STEC and ETEC in the diarrhoeic lambs implicated that these animals are important reservoir of STEC and ETEC. This is really a grave concern for the 'brokpas' and nomads (shepherds) who share a close relationship with this animals for their livelihood. This study also indicates that ETEC may be a major cause for frequent diarrhoeal episodes in lambs of this region.