Antibiotic Administration Routes and Oral Exposure to Antibiotic Resistant Bacteria as Key Drivers for Gut Microbiota Disruption and Resistome in Poultry.

Previous studies have identified oral administration of antibiotics and gut-impacting drugs as critical drivers for fecal antibiotic resistance (AR) and microbiome disruption in lab mice, but the practical implications of these findings have yet to be validated in hosts nurtured in conventional environment. Using ampicillin (Amp) as a way to extrapolate the general effect of antibiotics, this project examined the impact of drug administration routes on fecal microbiota and resistome using poultry raised in a teaching farm. AR genes were found to be abundant in the feces of young Leghorn chicks without previous antibiotic treatment. In chickens seeded with bla CMY-2 + Escherichia coli, 300 mg/kg body weight of Amp was orally administered for 5 days. This led to the fecal microbiota switching from Firmicutes occupied (95.60 ± 2.62%) and Lactobacillus rich, to being dominated by Proteobacteria (70.91 ± 28.93%), especially Escherichia/Shigella. However, when Amp was given via muscle injection, Firmicutes was mostly retained (i.e., from 83.6 ± 24.4% pre- to 90.4 ± 15.2% post-treatment). In control chickens without seeding with bla CMY-2 + E. coli, oral Amp also led to the increase of Proteobacteria, dominated by Klebsiella and Escherichia/Shigella, and a reduction of Firmicutes. Specifically within Firmicutes, Enterococcus, Clostridium, etc. were enriched but Lactobacillus was diminished. The fecal resistome including Ampr genes was more abundant in chickens receiving oral Amp than those treated with muscle injection, but the difference was primarily within 1 log. The data illustrated that both drug administration routes and pre-existing gut microbiota have profound impacts on gut microbiome disruption when antibiotic treatment is given. In hosts nurtured in a conventional environment, drug administration route has the most evident impact on gut microbiota rather than the size of the targeted bla CMY-2 + gene pool, likely due to the pre-existing bacteria that are (i) less susceptible to Amp, and/or (ii) with Ampr- or multidrug resistance-encoding genes other than bla CMY-2 +. These results demonstrated the critical interplay among drug administration routes, microbiota seeded through the gastrointestinal tract, AR, gut microbiota disruption, and the rise of common opportunistic pathogens in hosts. The potential implications in human and animal health are discussed.

Impact of Microbiota Transplant on Resistome of Gut Microbiota in Gnotobiotic Piglets and Human Subjects.

Microbiota transplant is becoming a popular process to restore or initiate "healthy" gut microbiota and immunity. But, the potential risks of the related practices need to be carefully evaluated. This study retrospectively examined the resistomes of donated fecal microbiota for treating intestinal disorders, vaginal microbiota of pregnant women, and infant fecal microbiota from rural and urban communities, as well as the impact of transplants on the fecal resistome of human and animal recipients. Antibiotic resistance (AR) genes were found to be abundant in all donor microbiota. An overall surge of resistomes with higher prevalence and abundance of AR genes was observed in the feces of all transplanted gnotobiotic pigs as well as in the feces of infant subjects, compared to those in donor fecal and maternal vaginal microbiota. Surprisingly, transplants using rural Amish microbiota led to more instead of less AR genes in the fecal microbiota of gnotobiotic pigs than did transplants using urban microbiota. New AR gene subtypes undetected originally also appeared in gnotobiotic pigs, in Crohn's Disease (CD) patients after transplant, and in feces of infant subjects. The data illustrated the key role of the host gastrointestinal tract system in amplifying the ever-increasing AR gene pool, even without antibiotic exposure. The data further suggest that the current approaches of microbiota transplant can introduce significant health risk factor(s) to the recipients, and newborn human and animal hosts with naïve gut microbiota were especially susceptible. Given the illustrated public health risks of microbiota transplant, minimizing massive and unnecessary damages to gut microbiota by oral antibiotics and other gut impacting drugs becomes important. Since eliminating risk factors including AR bacteria and opportunistic pathogens directly from donor microbiota is still difficult to achieve, developing microbial cocktails with defined organisms and functions has further become an urgent need, should microbiota transplantation become necessary.

Novel SCCmec type XII methicillin-resistant Staphylococcus aureus isolates identified from a swine production and processing chain.

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major public health concern. In this study, a total of 1485 samples from three swine farms, one slaughterhouse and one indoor market in Xiamen, China were collected in 2015, and the prevalence and profiles of MRSA were assessed. All the MRSA isolates were characterized by molecular typing, antibiotic susceptibility, coagulation activity, as well as PCR screening for 38 antibiotic resistance genes, two mobile genetic elements (lsa(E)-containing multiresistance gene cluster and Tn558), and 36 virulence genes. During the study, 54 of 1485 (3.6%) samples from the swine production, processing and retail chain were found positive for MRSA. A relatively rare SCCmecXII genotype was prevalent in swine farm (84.6%, 11/13) and slaughterhouse isolates (80.6%, 25/31), but absent in the market isolates (0%, 0/10). Notably, all staphylococcal cassette chromosome mec (SCCmec) type XII MRSA isolates were resistant to at least 6 classes of antibiotics, carried two mobile genetic elements (lsa(E)-containing multiresistance gene cluster and Tn558) and harbored multiple virulence genes. These multidrug resistant MRSA isolates could also coagulate both bovine and caprine plasma. Our results on the SCCmecXII MRSA isolates, particularly their profiles of related genotypes, antibiotic resistance and virulence determinants, illustrated the evolvement of livestock-associated (LA)-MRSA in the swine production environment and spread along the processing chain. The dominance of the SCCmecXII in MRSA isolates found in this study, differed from previous reports from China, indicated potential contribution associated with the production process.

Characterization of antibiotic resistance in commensal bacteria from an aquaculture ecosystem.

The objective of the study was to improve the understanding of antibiotic resistance (AR) ecology through characterization of antibiotic-resistant commensal isolates associated with an aquaculture production system. A total of 4767 isolates non-susceptible to sulfamethoxazole/trimethoprim (Sul/Tri), tetracycline (Tet), erythromycin (Erm), or cefotaxime (Ctx), originated from fish, feed, and environmental samples of an aquaculture farm with no known history of antibiotic applications were examined. Close to 80% of the isolates exhibited multi-drug resistance in media containing the corresponding antibiotics, and representative AR genes were detected in various isolates by PCR, with feed isolates had the highest positive rate detected. Identified AR gene carriers involved 18 bacterial genera. Selected AR genes led to acquired resistance in other bacteria by transformation. The AR traits in many isolates were stable in the absence of selective pressure. AR-rich feed and possibly environmental factors may contribute to AR in the aquaculture ecosystem. For minimum inhibitory concentration test, brain heart infusion medium was found more suitable for majority of the bacteria examined than cation-adjusted Mueller Hinton broth, with latter being the recommended medium for clinical isolates by standard protocol. The data indicated a need to update the methodology due to genetic diversity of microbiota for better understanding of the AR ecology.

Identification of a New Tetracycline Resistance Determinant tet47 from Fish Intestine.

To better understand food safety risks, functional genomic analysis was conducted to identify undescribed antibiotic resistance genes in fish samples from an aquaculture fish farm in Ohio. A fosmid genomic library from pooled DNA of antibiotic-resistant isolates was used to screen for resistance genes against tetracycline (Tet). A new Tet-resistant determinant designated as tet 47 was identified, with the original hosts being Providencia spp. from fish intestine. The new gene was also found to confer Tet resistance in Escherichia coli. Fish and byproducts were shown to be possible carriers that may disseminate new, functional, and potentially transmissible antibiotic resistance determinants through food, feed, and environmental contacts.

Role of a GntR-family response regulator LbrA in Listeria monocytogenes biofilm formation.

The formation of Listeria monocytogenes biofilms contributes to persistent contamination in food processing facilities. A microarray comparison of L. monocytogenes between the transcriptome of the strong biofilm forming strain (Bfm(s)) Scott A and the weak biofilm forming (Bfm(w)) strain F2365 was conducted to identify genes potentially involved in biofilm formation. Among 951 genes with significant difference in expression between the two strains, a GntR-family response regulator encoding gene (LMOf2365_0414), designated lbrA, was found to be highly expressed in Scott A relative to F2365. A Scott A lbrA-deletion mutant, designated AW3, formed biofilm to a much lesser extent as compared to the parent strain by a rapid attachment assay and scanning electron microscopy. Complementation with lbrA from Scott A restored the Bfm(s) phenotype in the AW3 derivative. A second microarray assessment using the lbrA deletion mutant AW3 and the wild type Scott A revealed a total of 304 genes with expression significantly different between the two strains, indicating the potential regulatory role of LbrA in L. monocytogenes. A cloned copy of Scott A lbrA was unable to confer enhanced biofilm forming potential in F2365, suggesting that additional factors contributed to weak biofilm formation by F2365.

Antibiotic administration routes significantly influence the levels of antibiotic resistance in gut microbiota.

This study examined the impact of oral exposure to antibiotic-resistant bacteria and antibiotic administration methods on antibiotic resistance (AR) gene pools and the profile of resistant bacteria in host gastrointestinal (GI) tracts using C57BL/6J mice with natural gut microbiota. Mice inoculated with a mixture of tet(M)-carrying Enterococcus spp. or blaCMY-2-carrying Escherichia coli were treated with different doses of tetracycline hydrochloride (Tet) or ampicillin sodium (Amp) and delivered via either feed or intravenous (i.v.) injection. Quantitative PCR assessment of mouse fecal samples revealed that (i) AR gene pools were below the detection limit in mice without prior inoculation of AR gene carriers regardless of subsequent exposure to corresponding antibiotics; (ii) oral exposure to high doses of Tet and Amp in mice inoculated with AR gene carriers led to rapid enrichment of corresponding AR gene pools in feces; (iii) significantly less or delayed development of AR in the GI tract of the AR carrier-inoculated mice was observed when the same doses of antibiotics were administered via i.v. injection rather than oral administration; and (iv) antibiotic dosage, and maybe the excretion route, affected AR in the GI tract. The shift of dominant AR bacterial populations in the gut microbiota was consistent with the dynamics of AR gene pools. The emergence of endogenous resistant bacteria in the gut microbiota corresponding to drug exposure was also observed. Together, these data suggest that oral administration of antibiotics has a prominent effect on AR amplification and development in gut microbiota, which may be minimized by alternative drug administration approaches, as illustrated by i.v. injection in this study and proper drug selection.

Antibiotic-resistant bacteria associated with retail aquaculture products from Guangzhou, China.

This study examined the prevalence of antibiotic-resistant (ART) bacteria and representative antibiotic resistance (AR)-encoding genes associated with several aquaculture products from retail markets in Guangzhou, China. ART commensal bacteria were found in 100% of the products examined. Among 505 multidrug-resistant isolates examined, close to one-fourth contained intI and sul1 genes: 15% contained sul2 and 5% contained tet (E). Incidences of ß-lactamase-encoding genes bla(TEM), bla(CMY) and erythromycin resistance determinants ermB and ermC were 4.5, 1.7, 1.3, and 0.3%, respectively. Most of the ART isolates identified from the rinse water were Aeromonas spp.; those from intestines belonged to the Enterobacteriaceae. Plasmid-associated intI and AR-encoding genes were identified in several ART isolates by Southern hybridization. Three multidrug resistance-encoding plasmids were transferred into Escherichia coli DH5 a by chemical transformation and led to acquired AR in the transformants. In addition, the AR traits in many isolates were quite stable, even in the absence of selective pressure. Further studies are needed to reveal risk factors associated with the aquaculture production chain for targeted AR mitigation.

Critical issues in detecting viable Listeria monocytogenes cells by real-time reverse transcriptase PCR.

Rapid and specific detection of viable Listeria monocytogenes cells, particularly in processed foods, is a major challenge in the food industry. To assess the suitability of using RNA-based detection methods to detect viable cells, several sets of PCR primers and florescent probes were designed targeting the 16S rRNA, internalin A, and ribosomal protein L4 genes. One-step real-time reverse transcriptase (RT) PCR assays were conducted using RNAs extracted from control and heat-treated L. monocytogenes samples. The cycle threshold values were significantly higher in heat-treated cells than in controls. However, real-time RT-PCR amplification signals were still detected even in samples stored at room temperature for 24 h after lethal treatments, and the intensity of the signals was correlated with the cell population. The 16S rRNA molecules were the most stable of the three targets evaluated, and the impact on detection efficacy of the relative positions of the PCR primers within the target genes was limited under the experimental conditions. These results suggest that real-time RT-PCR assays have advantages over conventional PCR assays for assessing viable L. monocytogenes cells, but the results are affected by the stability of the RNA molecules targeted. These findings could have a major impact on interpretation of RNA-based detection data and gene expression studies.

Acquired antibiotic resistance: are we born with it?

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including tet(M), ermB, sul2, and bla(TEM) were detected in infant subjects. Enterococcus spp., Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Escherichia coli/Shigella spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.

Persistent, toxin-antitoxin system-independent, tetracycline resistance-encoding plasmid from a dairy Enterococcus faecium isolate.

A tetracycline-resistant (Tet(r)) dairy Enterococcus faecium isolate designated M7M2 was found to carry both tet(M) and tet(L) genes on a 19.6-kb plasmid. After consecutive transfer in the absence of tetracycline, the resistance-encoding plasmid persisted in 99% of the progenies. DNA sequence analysis revealed that the 19.6-kb plasmid contained 28 open reading frames (ORFs), including a tet(M)-tet(L)-mob gene cluster, as well as a 10.6-kb backbone highly homologous (99.9%) to the reported plasmid pRE25, but without an identified toxin-antitoxin (TA) plasmid stabilization system. The derived backbone plasmid without the Tet(r) determinants exhibited a 100% retention rate in the presence of acridine orange, suggesting the presence of a TA-independent plasmid stabilization mechanism, with its impact on the persistence of a broad spectrum of resistance-encoding traits still to be elucidated. The tet(M)-tet(L) gene cluster from M7M2 was functional and transmissible and led to acquired resistance in Enterococcus faecalis OG1RF by electroporation and in Streptococcus mutans UA159 by natural transformation. Southern hybridization showed that both the tet(M) and tet(L) genes were integrated into the chromosome of S. mutans UA159, while the whole plasmid was transferred to and retained in E. faecalis OG1RF. Quantitative real-time reverse transcription-PCR (RT-PCR) indicated tetracycline-induced transcription of both the tet(M) and tet(L) genes of pM7M2. The results indicated that multiple mechanisms might have contributed to the persistence of antibiotic resistance-encoding genes and that the plasmids pM7M2, pIP816, and pRE25 are likely correlated evolutionarily.

Effective antibiotic resistance mitigation during cheese fermentation.

Controlling antibiotic-resistant (ART) bacteria in cheese fermentation is important for food safety and public health. A plant-maintained culture was found to be a potential source for ART bacterial contamination in cheese fermentation. Antibiotics had a detectable effect on the ART population from contamination in the finished product. The decrease in the prevalence of antibiotic resistance (AR) in retail cheese samples from 2010 compared to data from 2006 suggested the effectiveness of targeted AR mitigation in related products.

Tetracycline resistance associated with commensal bacteria from representative ready-to-consume deli and restaurant foods.

Proper knowledge of antibiotic resistance (AR) dissemination is essential for effective mitigation. This study examined the profiles of tetracycline-resistant (Tetr) commensal bacteria from representative ready-to-consume food samples from salad bars at local grocery stores and restaurants. Out of 900 Tetr isolates examined, 158 (17.6%) carried one or more of tetM, tetL, tetS, and tetK genes by conventional PCR, 28 harbored more than one Tetr determinants. The most prevalent genotype was tetM, which was detected in 70.9% of the AR gene carriers, followed by tetL (31.6%), tetS (13.9%), and tetK (2.5%). Identified AR gene carriers included Enterococcus, Lactococcus, Staphylococcus, Brochothrix, Carnobacterium, Stenotrophomonas, Pseudomonas, and Sphingobacterium, by 16S rRNA gene sequence analysis. AR determinants were successfully transmitted, and led to resistance in Streptococcus mutans via natural gene transformation and Enterococcus faecalis via electroporation, suggesting the functionality and mobility of the AR genes from the food commensal bacteria. In addition, the AR traits in many isolates are quite stable, even in the absence of the selective pressure. The identification of new commensal carriers for representative AR genes revealed the involvement of a broad spectrum of bacteria in the horizontal transmission of AR genes. Meanwhile, the spectrum of the antibiotic-resistant bacteria differed from the spectrum of the total bacteria (by denaturing gradient gel electrophoresis) associated with the food items. Our data revealed a common avenue in AR exposure and will assist in proper risk assessment and the development of comprehensive mitigation strategies to effectively combat AR.

Quantitative assessment of the tetracycline resistance gene pool in cheese samples by real-time TaqMan PCR.

A TaqMan real-time PCR assay was developed to quantify the tetS gene pool present in retail cheeses. This protocol offers a rapid, specific, sensitive, and culture-independent method for assessing antibiotic resistance genes in food samples rich in fats and proteins.

Effect of passive cigarette smoking on colonic alpha7-nicotinic acetylcholine receptors in TNBS-induced colitis in rats.

BACKGROUND AND AIMS: Cigarette smoking affects colonic inflammation, but the exact mechanism by which it does so is unclear. The aim of this study was to investigate the underlying mechanism by examining the effect of cigarette smoking on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. METHOD: Experimental colitis was induced by administrating TNBS enema in Sprague-Dawley rats. The effect of cigarette smoking was assessed by measuring the colonic edema, mucosal lesion area, histopathological score, mucosal myeloperoxidase (MPO) activity and tumor necrosis factor-alpha (TNF-alpha). The expression of alpha7-nicotinic acetylcholine receptor (alpha7nAChR) was examined after cigarette smoking to identify whether the alpha7nAChR is involved in inflammation. RESULTS: TNBS induced severe colitis as evidenced by increased colonic edema, mucosal lesion area, histopathological score, MPO activity, and TNF-alpha level. Inflammation was aggravated by cigarette smoke exposure. Colonic tissue expressed alpha7nAChR (0.41 +/- 0.11), and TNBS up-regulated the receptor expression (0.46 +/- 0.11), but the difference was not significant (p > 0.05). Cigarette smoking with 2 and 4% respectively significantly increased the expression of alpha7nAChR (0.55 +/- 0.05 and 0.64 +/- 0.08) (p < 0.05). CONCLUSIONS: The up-regulated expression of alpha7nAChR after exposure to cigarette smoking indicates that alpha7nAChR in colonic tissue may be involved in cigarette smoking damage in TNBS-induced colitis in rats.

Assessment of environmental factors on Listeria monocytogenes Scott A inlA gene expression by relative quantitative Taqman real-time reverse transcriptase PCR.

Several virulence factors are involved in Listeria monocytogenes pathogenicity. L. monocytogenes internalins, particularly internalin A, are required for bacterial adhesion to and invasion of human intestinal epithelial cells. The expression of internalins is thus related to virulence. Identification of conditions involved in regulating the expression of L. monocytogenes virulence factors is essential for developing targeted strategies to control listeriosis incidence and improving therapeutic approaches. The primary aim of this study was to develop a quantitative real-time reverse transcriptase PCR platform to study the impact of environmental factors on L. monocytogenes Scott A virulence factor expression, particularly in potentially complex ecosystems. A Taqman PCR-based, rapid quantitative gene expression evaluation method was established with the L. monocytogenes ribosomal protein L4 encoding gene used as an internal standard. Our data suggest that inlA expression is influenced by food composition and temperature, indicating that certain food processing or storage conditions, such as the use of lactic and acetic acids at common storage temperatures, could affect the expression of L. monocytogenes virulence factor.

Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes.

The rapid emergence of antibiotic-resistant (ART) pathogens is a major threat to public health. While the surfacing of ART food-borne pathogens is alarming, the magnitude of the antibiotic resistance (AR) gene pool in food-borne commensal microbes is yet to be revealed. Incidence of ART commensals in selected retail food products was examined in this study. The presence of 10(2)-10(7) CFU of ART bacteria per gram of foods in many samples, particularly in ready-to-eat, 'healthy' food items, indicates that the ART bacteria are abundant in the food chain. AR-encoding genes were detected in ART isolates, and Streptococcus thermophilus was found to be a major host for AR genes in cheese microbiota. Lactococcus lactis and Leuconostoc sp. isolates were also found carrying AR genes. The data indicate that food could be an important avenue for ART bacterial evolution and dissemination. AR-encoding plasmids from several food-borne commensals were transmitted to Streptococcus mutans via natural gene transformation under laboratory conditions, suggesting the possible transfer of AR genes from food commensals to human residential bacteria via horizontal gene transfer.

Rapid, specific, and sensitive detection of spoilage molds in orange juice using a real-time Taqman PCR assay.

The outgrowth of spoilage organisms, including molds and yeasts, results in significant financial loss to the food industry and wastes natural resources. The objective of this study was to develop a rapid, specific, and sensitive real-time PCR method for detecting spoilage molds during screening of raw materials and final product quality control analysis. The 18S rRNA gene was used to develop PCR primers and probe. With this set of primers and probe, less than 1,000 mold cells per milliliter of orange juice (10 cells per reaction) were detected with the real-time PCR system within 6 to 7 h. No cross-reactivity was found with other common foodborne bacteria, yeasts, or food ingredients. This technique is significantly faster than current detection and identification procedures, which take from days to weeks.

High-frequency conjugation system facilitates biofilm formation and pAMbeta1 transmission by Lactococcus lactis.

The importance of conjugation as a mechanism to spread biofilm determinants among microbial populations was illustrated with the gram-positive bacterium Lactococcus lactis. Conjugation triggered the enhanced expression of the clumping protein CluA, which is a main biofilm attribute in lactococci. Clumping transconjugants further transmitted the biofilm-forming elements among the lactococcal population at a much higher frequency than the parental non-clumping donor. This cell-clumping-associated high-frequency conjugation system also appeared to serve as an internal enhancer facilitating the dissemination of the broad-host-range drug resistance gene-encoding plasmid pAMbeta1 within L. lactis, at frequencies more than 10,000 times higher than those for the non-clumping parental donor strain. The implications of this finding for antibiotic resistance gene dissemination are discussed.