Hua H. Wang

Antibiotic Administration Routes Significantly Influence the Levels of Antibiotic Resistance in Gut Microbiota

ABSTRACT 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.

Biofilms in the Food Environment

Biofilms are structured microbial communities of cells differentiated to play specific roles in the maintenance of the community and its structure. In this chapter, the discussion of biofilm development is organized into three sections: initiation (reversible and irreversible attachment), structure development (maturation), and dispersal. Although there is a general process for how biofilms develop, mechanisms underlying the process differ among microorganisms. The chapter talks about the biofilm matrix, and biofilm ecosystem. Research on food processing biofilms has centered on the ability of pathogenic and spoilage microorganisms to grow or survive in these environments, with emphasis on the influence of sanitation procedures. Researchers isolated two types of rough colony variants of Listeria monocytogenes from biofilms, distinguished by short-chain and long-chain cell morphologies. Both types of rough variants exhibited enhanced biofilm formation, with the variants exhibiting increased cell chain length (filamentous growth) when grown as biofilms. The predominant microflora of water system biofilms can be characterized as having low physiological activity and as being difficult to culture using conventional plating methods. Biofilms containing mainly commensal microorganisms can form on roots, leaves, and the internal vascular tissues of edible plants. Cells in biofilms are more difficult to inactivate by application of antimicrobial chemicals and physical stresses than their planktonic counterparts. The major contributor of biofilm microorganisms in our diet is most likely fresh produce, since biofilms form on these foods before harvest, postharvest growth is likely, and the products are consumed without heat treatment.

Antibiotic Resistance: How Much Do We Know and Where Do We Go from Here?

The rapid dissemination of antibiotic resistant (ART) pathogens threatens human health and may have significant social and financial impacts. It is well recognized that applications of antibiotics in human clinical therapy, aquaculture, and food animal production all contribute to the emergence and

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

ABSTRACT A tetracycline-resistant (Tetr) 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 Tetr 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.

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.

Quantitative Assessment of the Tetracycline Resistance Gene Pool in Cheese Samples by Real-Time TaqMan PCR

ABSTRACT 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.

The mosaic accessory gene structures of the SXT/R391-like integrative and conjugative elements derived from Vibrio spp. isolated from aquatic products and environment in the Yangtze River estuary, China

BackgroundThe emergence, resurgence and spread of human food-borne pathogenic Vibrios are one of the major contributors to disease burden and mortality particularly in developing countries with disputable sanitary conditions. Previous research on pathogenic Vibrio cholerae and Vibrio parahaemolitycus derived from clinical samples has proposed links between acquisition of virulence and multiple drug resistance traits and intercellular transmissibility of mobile genetic elements in the environment. To date, very few information is available on environmental Vibrio isolates. In this study, we characterized eleven Vibrio strains bearing the SXT/R391-like integrative and conjugative elements (ICEs) derived from aquatic products and environment in the Yangtze River Estuary, China.ResultsThe eleven Vibrio strains were isolated in 2010 to 2011, and taxonomically identified, which included six Vibrio cholerae, three Vibrio parahaemolyticus, one Vibrio alginolyticus and one Vibrio natriegens. Most of the strains displayed strong resistance phenotypes to ampicillin, mercury and chromium. The majority of their ICEs, which belong to S and R exclusion system groups, contain ICEs-chromosome junction sequences and highly conserved core-genes required for ICE transfer. However, comparative sequence analysis uncovered interesting diversity in their mosaic accessory gene structures, which carry many novel genes that have not been described in any known ICEs to date. In addition, antibiotic resistance was transmitted by ICEVchChn6 and ICEVpaChn1 from V. cholerae, V. parahaemolyticus to E. coli MG1655 via conjugation, respectively. Our data also revealed that the ICEs characterized in this study are phylogenetically distant from most of the SXT/R391 ICEs reported previously, which may represent a novel cluster likely shaped by the ecological environment in the Yangtze River Estuary, China.ConclusionsThis study constitutes the first investigation of ICEs-positive Vibrio spp. in the Yangze River Estuary, China. The newly identified ICEs were characterized with mosaic accessory gene structures and many novel genes. The results demonstrated self-transmissibility of antibiotic resistance mediated by two of the ICEs from V. cholerae, V. parahaemolyticus to E. coli via conjugation, respectively. Our results also revealed that the ICEs examined in this study may represent a novel cluster in the SXT/R391 family.

New variant Salmonella genomic island 1-U in Proteus mirabilis clinical and food isolates from South China

Sir, Salmonella genomic island 1 (SGI1) is a 43 kb genomic island containing a 13 kb multidrug resistance (MDR) region antibiotic resistance gene cluster initially identified in Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 strains. Classically, the MDR region contains five antibiotic resistance genes, all of which are located within the boundaries of a complex class 1 integron, designated In104. Over the past few years, SGI1 with variant antibiotic resistance gene clusters (termed variable SGI1) named SGI1-A to SGI1-T have been identified in several other Salmonella serotypes and also in Proteus mirabilis, – 4 suggesting possible horizontal gene transmission of the genetic element among microbes. However, it is worth noting that the direct association of the int2 gene, which is part of a retron sequence, with SGI1 has been reported only in S. enterica Typhimurium, while SGI1 is located between the thdF and yidY genes in other S. enterica serovars. The integration of SGI1 into the P. mirabilis chromosome takes place between the thdF and hipB genes, according to the report by Boyd et al. However, our overall understanding of the evolution of SGI1 and the potential risk of its dissemination among microbes remains very limited. To reveal the genetic difference between SGI1 variants and the potential molecular events involved in the evolution and transmission of the MDR-encoding unit, based on an identical MDR profile to sulfamethoxazole, trimethoprim, tetracycline and erythromycin, a total of 23 P. mirabilis strains isolated in Guangzhou, China, were examined in this study. Seventeen of them originated from stools of 17 patients with diarrhoea in the First Affiliated Hospital of Jinan University from 2006 to 2008. The remaining six isolates were from 35 raw chicken meat samples purchased from two markets in 2009. PCR primers used in the screening for SGI1 are listed in Table S1 (available as Supplementary data at JAC Online). Eight P. mirabilis strains including three from chicken samples from the same seller in one of the markets and the other five from patients from 2006 to 2008 were found to harbour the dfrA5 cassette of class 1 integrons. To confirm the presence of SGI1, primer pairs corresponding to the left junction region (PmLJ1/LJ-R1) and right junction region (104-RJ/PmRJ1) of SGI1 in the P. mirabilis chromosome were used. PCR results were positive for the left junction of SGI1 with the thdF gene, but negative for the right junction with the hipB gene. The right junction region of SGI1 was then analysed by PCR with primers hipA-R1 (hipA specific) and MP-R1 (specific for the membrane protein PMI3124-encoding gene) targeting the two genes downstream of hipB of P. mirabilis HI4320. Positive results were found in all eight strains using primer pair 104-RJ and MP-R1. The PCR products were sequenced and the sequences flanking SGI1 were, with the exception of a few base changes, identical to part of the S044and PMI3124-encoding genes. Thus, the eight P. mirabilis strains appeared to have SGI1 or variants integrated between thdF and the gene encoding PMI3124. Besides, the same integration site has been reported in a P. mirabilis isolate from France containing the SGI1-O variant. Interestingly, the hipB and hipA genes were further screened using specific primers hipB-F1 and hipA-R1, and the results were negative. The results indicated that the eight P. mirabilis strains lacked the sequences of hipB and hipA genes (Figure 1). PCR mapping of the antibiotic resistance gene cluster was performed using primers described in Table S1. PCR cloning and DNA sequencing revealed that all eight strains harboured a new variant designated SGI1-U. It contained the dfrA5 gene cassette within the integron structure. It is noteworthy that the dfrA5 cassette coding for trimethoprim resistance had not been reported in P. mirabilis before. Thus, the strains probably obtained the dfrA5 gene cassette from other microorganisms. The three strains from food and the five strains from patients had 100% identical SfiI and NotI PFGE patterns, suggesting that they might be clonally related. In conclusion, for the first time, the new variant SGI1-U was identified in eight P. mirabilis clinical and food isolates from South China. It is also interesting that this is the first time the dfrA5 gene has been identified in P. mirabilis. In particular, the deletions of hipB and hipA genes found at the right junction region of SGI1-U showed that SGI1-U was located between the thdF gene and a gene encoding a membrane protein on the P. mirabilis chromosome.

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 β-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.

Comparative secretomics reveals novel virulence-associated factors of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a causative agent of serious human seafood-borne gastroenteritis disease and even death. In this study, for the first time, we obtained the secretomic profiles of seven V. parahaemolyticus strains of clinical and food origins. The strains exhibited various toxic genotypes and phenotypes of antimicrobial susceptibility and heavy metal resistance, five of which were isolated from aquatic products in Shanghai, China. Fourteen common extracellular proteins were identified from the distinct secretomic profiles using the two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) techniques. Of these, half were involved in protein synthesis and sugar transport of V. parahaemolyticus. Strikingly, six identified proteins were virulence-associated factors involved in the pathogenicity of some other pathogenic bacteria, including the translation elongation factor EF-Tu, pyridoxine 5′-phosphate synthase, σ54 modulation protein, dihydrolipoyl dehydrogenase, transaldolase and phosphoglycerate kinase. In addition, comparative secretomics also revealed several extracellular proteins that have not been described in any bacteria, such as the ribosome-recycling factor, translation elongation factor EF-Ts, phosphocarrier protein HPr and maltose-binding protein MalE. The results in this study will facilitate the better understanding of the pathogenesis of V. parahaemolyticus and provide data in support of novel vaccine candidates against the leading seafood-borne pathogen worldwide.