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1.  Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits 
Applied and Environmental Microbiology  2014;80(12):3562-3567.
Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.
PMCID: PMC4054130  PMID: 24705326
2.  Mortality in Kittens Is Associated with a Shift in Ileum Mucosa-Associated Enterococci from Enterococcus hirae to Biofilm-Forming Enterococcus faecalis and Adherent Escherichia coli 
Journal of Clinical Microbiology  2013;51(11):3567-3578.
Approximately 15% of foster kittens die before 8 weeks of age, with most of these kittens demonstrating clinical signs or postmortem evidence of enteritis. While a specific cause of enteritis is not determined in most cases, these kittens are often empirically administered probiotics that contain enterococci. The enterococci are members of the commensal intestinal microbiota but also can function as opportunistic pathogens. Given the complicated role of enterococci in health and disease, it would be valuable to better understand what constitutes a “healthy” enterococcal community in these kittens and how this microbiota is impacted by severe illness. In this study, we characterized the ileum mucosa-associated enterococcal community of 50 apparently healthy and 50 terminally ill foster kittens. In healthy kittens, Enterococcus hirae was the most common species of ileum mucosa-associated enterococci and was often observed to adhere extensively to the small intestinal epithelium. These E. hirae isolates generally lacked virulence traits. In contrast, non-E. hirae enterococci, notably Enterococcus faecalis, were more commonly isolated from the ileum mucosa of kittens with terminal illness. Isolates of E. faecalis had numerous virulence traits and multiple antimicrobial resistances. Moreover, the attachment of Escherichia coli to the intestinal epithelium was significantly associated with terminal illness and was not observed in any kitten with adherent E. hirae. These findings identify a significant difference in the species of enterococci cultured from the ileum mucosa of kittens with terminal illness compared to the species cultured from healthy kittens. In contrast to prior case studies that associated enteroadherent E. hirae with diarrhea in young animals, these controlled studies identified E. hirae as more often isolated from healthy kittens and adherence of E. hirae as more common and extensive in healthy kittens than in sick kittens.
PMCID: PMC3889735  PMID: 23966487
3.  Evaluation of Post Partum Depression in a Tertiary Hospital 
To evaluate the association of different factors with postpartum depression.
A prospective study conducted in the Department of Obstetrics and Gynecology, Medical College, Kolkata. Six thousand patients, 4–7 days postpartum, were interrogated using Edinburgh Postnatal Depression Scale (EPDS). Sociodemographic factors (age, parity, literacy, socioeconomic status, marital status and family structure), history of psychiatric disorder and abuse, mode of delivery and obstetric outcome were recorded. The results were analyzed statistically using Chi-square test.
Incidence of PPD was 25%. Significant association of PPD was seen with poor socioeconomic group (P < 0.05), literacy (P < 0.001), nuclear family structure (P < 0.05), single mother (P < 0.001), past history of psychiatric illness (P < 0.001), history of abuse (P < 0.05), and poor obstetric outcome (P < 0.001). Age, parity and method of delivery showed no association.
EPDS should be used routinely to screen for PPD among high risk cases.
PMCID: PMC3257346  PMID: 23024522
Postnatal depression; Edinburgh postnatal depression scale
4.  Branching of the p-nitrophenol (PNP) degradation pathway in burkholderia sp. Strain SJ98: Evidences from genetic characterization of PNP gene cluster 
AMB Express  2012;2:30.
Aerobic microbial degradation of p-nitrophenol (PNP) has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative bacteria, whereas in Gram-positive PNP degraders it proceed via ‘Benzenetriol (BT) pathway’. These pathways are characterized by the ring cleavage of HQ and BT as terminal aromatic intermediates respectively. Earlier reports on PNP degradation have indicated these pathways to be mutually exclusive. We report involvement of both ‘HQ’ and ‘BT’ ring cleavage pathways in PNP degradation by Burkholderia sp. strain SJ98. Genetic characterization of an ~41 Kb DNA fragment harboring PNP degradation gene cluster cloned and sequenced from strain SJ98 showed presence of multiple orfs including pnpC and pnpD which corresponded to previously characterized ‘benzenetriol-dioxygenase (BtD)’ and ‘maleylacetate reductase (MaR)’ respectively. This gene cluster also showed presence of pnpE1 and pnpE2, which shared strong sequence identity to cognate sub-units of ‘hydroquinone dioxygenase’ (HqD). Heterologous expression and biochemical characterization ascertained the identity of PnpE1 and PnpE2. In in vitro assay reconstituted heterotetrameric complex of PnpE1 and PnpE2 catalyzed transformation of hydroquinone (HQ) into corresponding hydroxymuconic semialdehyde (HMS) in a substrate specific manner. Together, these results clearly establish branching of PNP degradation in strain SJ98. We propose that strain SJ98 presents a useful model system for future studies on evolution of microbial degradation of PNP.
PMCID: PMC3485097  PMID: 22681853
P-nitrophenol; Hydroquinone dioxygenase; PNP pathway; Burkholderia sp SJ98
5.  Resident Cats in Small Animal Veterinary Hospitals Carry Multi-Drug Resistant Enterococci and are Likely Involved in Cross-Contamination of the Hospital Environment 
In the USA, small animal veterinary hospitals (SAVHs) commonly keep resident cats living permanently as pets within their facilities. Previously, multi-drug resistant (MDR) enterococci were found as a contaminant of multiple surfaces within such veterinary hospitals, and nosocomial infections are a concern. The objectives of this study were to determine whether resident cats carry MDR enterococci and to compare the feline isolates genotypically to those obtained from SAVH surfaces in a previous study. Enterococcal strains (n = 180) were isolated from the feces of six healthy resident cats from different SAVHs. The concentration of enterococci ranged from 1.1 × 105 to 6.0 × 108 CFU g−1 of feces, and the population comprised Enterococcus hirae (38.3 ± 18.6%), E. faecium (35.0 ± 14.3%), E. faecalis (23.9 ± 11.0%), and E. avium (2.8 ± 2.2%). Testing of phenotypic resistance to 14 antimicrobial agents revealed multi-drug resistance (≥3 antimicrobials) in 48.9% of all enterococcal isolates with most frequent resistance to tetracycline (75.0%), erythromycin (50.0%), and rifampicin (36.1%). Vancomycin resistant E. faecalis (3.9%) with vanB not horizontally transferable in in vitro conjugation assays were detected from one cat. Genotyping with pulsed-field gel electrophoresis demonstrated a host-specific clonal population of MDR E. faecalis and E. faecium. Importantly, several feline isolates were genotypically identical or closely related to isolates from surfaces of cage door, thermometer, and stethoscope of the corresponding SAVHs. These data demonstrate that healthy resident cats at SAVHs carry MDR enterococci and likely contribute to contamination of the SAVH environment. Proper disposal and handling of fecal material and restricted movement of resident cats within the ward are recommended.
PMCID: PMC3282945  PMID: 22363334
Enterococcus; antimicrobial resistance; resident cats; small animal veterinary hospital; clonal diversity
6.  Chemotaxis of Burkholderia sp. Strain SJ98 towards chloronitroaromatic compounds that it can metabolise 
BMC Microbiology  2012;12:19.
Burkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2-chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds.
Strain SJ98 could mineralize 2C4NP, 4C2NB and 5C2NB, and co-metabolically transform 2C3NP and 2C4NB in the presence of an alternative carbon source, but was unable to transform 4C2NP under these conditions. Positive chemotaxis was only observed towards the five metabolically transformed CNACs. Moreover, the chemotaxis was induced by growth in the presence of the metabolisable CNAC. It was also competitively inhibited by the presence of nitroaromatic compounds (NACs) that it could metabolise but not by succinate or aspartate.
Burkholderia sp. strain SJ98 exhibits metabolic transformation of, and inducible chemotaxis towards CNACs. Its chemotactic responses towards these compounds are related to its previously demonstrated chemotaxis towards NACs that it can metabolise, but it is independently inducible from its chemotaxis towards succinate or aspartate.
PMCID: PMC3293717  PMID: 22292983
7.  Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer 
PLoS ONE  2011;6(7):e22451.
The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks.
PMCID: PMC3139645  PMID: 21811613
8.  Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community 
BMC Microbiology  2011;11:23.
Extensive use of antibiotics as growth promoters in the livestock industry constitutes strong selection pressure for evolution and selection of antibiotic resistant bacterial strains. Unfortunately, the microbial ecology and spread of these bacteria in the agricultural, urban, and suburban environments are poorly understood. Insects such as house flies (Musca domestica) and German cockroaches (Blattella germanica) can move freely between animal waste and food and may play a significant role in the dissemination of antibiotic resistant bacteria within and between animal production farms and from farms to residential settings.
Enterococci from the digestive tract of house flies (n = 162), and feces of German cockroaches (n = 83) and pigs (n = 119), collected from two commercial swine farms were isolated, quantified, identified, and screened for antibiotic resistance and virulence. The majority of samples (93.7%) were positive for enterococci with concentrations 4.2 ± 0.7 × 104 CFU/house fly, 5.5 ± 1.1 × 106 CFU/g of cockroach feces, and 3.2 ± 0.8 × 105 CFU/g of pig feces. Among all the identified isolates (n = 639) Enterococcus faecalis was the most common (55.5%), followed by E. hirae (24.9%), E. faecium (12.8%), and E. casseliflavus (6.7%). E. faecalis was most prevalent in house flies and cockroaches, and E. hirae was most common in pig feces. Our data showed that multi-drug (mainly tetracycline and erythromycin) resistant enterococci were common from all three sources and frequently carried antibiotic resistance genes including tet(M) and erm(B) and Tn916/1545 transposon family. E. faecalis frequently harbored virulence factors gelE, esp, and asa1. PFGE analysis of selected E. faecalis and E. faecium isolates demonstrated that cockroaches and house flies shared some of the same enterococcal clones that were detected in the swine manure indicating that insects acquired enterococci from swine manure.
This study shows that house flies and German cockroaches in the confined swine production environment likely serve as vectors and/or reservoirs of antibiotic resistant and potentially virulent enterococci and consequently may play an important role in animal and public health.
PMCID: PMC3039560  PMID: 21269466
9.  Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria 
Indian Journal of Microbiology  2008;48(2):279-286.
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.
PMCID: PMC3450173  PMID: 23100721
Benzenetriol dioxygenase; p-Nitrophenol; Phylogenetic analysis

Results 1-9 (9)