Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.     

Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 10³ CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.     

Prevalence of Virulence Genes and Antibiotic Resistance Pattern in Enterococcus Faecalis Isolated from Urinary Tract Infection in Shahrekord,Iran

Background:This study aims to specify the antimicrobial resistance pattern and virulence genes of Enterococcus faecalis isolated from urinary tract infections in Shahrekord, Iran. Methods:Urine samples of 1000 people suspected of having urinary tract infections referred to Shahrekord medical diagnostic laboratories were examined. Biofilm assays were performed by microtiter plate test through reading the OD490. Polymerase Chain Reaction (PCR) was applied to study the virulence factors.Results:Enterococcus faecalis was detected in 60 samples. After performing microbiological tests, all samples were positive in the molecular analysis. Strong, moderate and weak biofilm reactions reported 66.67%, 25%, and 8.33% respectively. The most resistance reported to cotrimoxazole, vancomycin and amikacin and the lowest resistance to nitrofurantoin (8.33%) was reported. Statistical analysis with Fisher''s exact test showed a statistically significant relationship between biofilm production and resistance to cotrimoxazole, vancomycin and cefotaxime. Prevalence of efe A, ace, gel E, esp, cyl M, agg, cyl A and cyl B in strong biofilm formation isolates was reported 100%, 87.5%, 82%, 62.5%, 55%, 37.5% 25% and 22.5% respectively. There was a significant relationship between the frequency of efa A and strong biofilm reaction.Conclusion:The presence of E. faecalis strains resistant to co-trimoxazole and vancomycin and present of some virulence factors is alarming the researchers. Since antibiotic resistance genes are probably transmitted among enterococci, and Staphylococci, controlling infections made by enterococci as well as the appropriate administration of antibiotics could treat the nosocomial infections effectively.Key Words: Antibiotic Resistance, Enterococcus faecalis, Urinary Tract Infection, Virulence genes     

Mechanism of Resistance to Antibiotic Synergism in Enterococci

Enterococci exhibit two types of resistance to streptomycin. Moderately high-level resistance is observed in most naturally occurring strains and can be overcome by simultaneous exposure to penicillin. In addition, very high-level resistance is found in those strains against which penicillin plus streptomycin fail to produce synergism in vitro. To study the mechanism of streptomycin resistance in enterococci, ribosomes from a wild-type strain and from a highly streptomycin-resistant mutant were isolated, characterized, and studied in an in vitro amino acid incorporation system. The ribosomes from the organism with moderately high-level streptomycin resistance were sensitive to streptomycin in vitro, suggesting that this type of resistance is caused by failure of streptomycin to reach the ribosomes. Very high-level resistance (and lack of penicillin-streptomycin synergism), on the other hand, appears to be due to ribosomally mediated streptomycin resistance.     

Frequency of blaCTX-M and blaTEM Virulence Genes and Antibiotic Resistance Profiles among Klebsiella pneumoniae Isolates in Urinary Tract Infection (UTI) Samples from Hashtgerd,Iran

Background:Klebsiella pneumoniae (K. pneumoniae) is an opportunistic microorganism and one of the most important causes of urinary tract infection. This study aimed to evaluate the frequency of K. pneumoniae producing broad-spectrum beta-lactamase in urinary tract infection and to determine the pattern of drug resistance.Methods:This study was performed on 50 samples of K. pneumoniae isolated from patients with urinary tract infection referred to the Medical Diagnostic Laboratory in Hashtgerd city. The isolates were first evaluated for antibiotic susceptibility by disk diffusion method according to the method proposed by the Clinical and Laboratory Standards Institute (CLSI). Then phenotypic detection of ESBLS was carried out by the DDST method. The frequency of gene bla_TEM and bla_CTX-M was determined by PCR.Results:The highest resistance was observed to ampicillin (94%) and the highest sensitivity was observed to gentamicin (84%). 22 isolates (44%) were positive for ESBLs production. Of the 50 isolates studied, 34% had bla_CTX-M and 28% had bla_TEM and 11 (22%) had both genes simultaneously. Also, more than 77% of positive ESBLs isolates had the bla_CTX-M gene and approximately 63.64% of positive ESBLs isolates had the bla_TEM gene.Conclusion:Given the high prevalence of antibiotic-resistant and ESBL-producing isolates, early identification of these resistant isolates and their follow-up is essential to prevent further outbreaks. It is also important to use appropriate therapeutic strategies and proper and rational administration of antibiotics by physicians.Key Words: Antibiotic Resistance, ESBLs, K. pneumoniae, Urinary Tract Infection     

Resistance Genes and Genetic Elements Associated with Antibiotic Resistance in Clinical and Commensal Isolates of Streptococcus salivarius

The diversity of clinical (n = 92) and oral and digestive commensal (n = 120) isolates of Streptococcus salivarius was analyzed by multilocus sequence typing (MLST). No clustering of clinical or commensal strains can be observed in the phylogenetic tree. Selected strains (92 clinical and 46 commensal strains) were then examined for their susceptibilities to tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicol antibiotics. The presence of resistance genes tet(M), tet(O), erm(A), erm(B), mef(A/E), and catQ and associated genetic elements was investigated by PCR, as was the genetic linkage of resistance genes. High rates of erythromycin and tetracycline resistance were observed among the strains. Clinical strains displayed either the erm(B) (macrolide-lincosamide-streptogramin B [MLS_B] phenotype) or mef(A/E) (M phenotype) resistance determinant, whereas almost all the commensal strains harbored the mef(A/E) resistance gene, carried by a macrolide efflux genetic assembly (MEGA) element. A genetic linkage between a macrolide resistance gene and genes of Tn916 was detected in 23 clinical strains and 5 commensal strains, with a predominance of Tn3872 elements (n = 13), followed by Tn6002 (n = 11) and Tn2009 (n = 4) elements. Four strains harboring a mef(A/E) gene were also resistant to chloramphenicol and carried a catQ gene. Sequencing of the genome of one of these strains revealed that these genes colocalized on an IQ-like element, as already described for other viridans group streptococci. ICESt3-related elements were also detected in half of the isolates. This work highlights the potential role of S. salivarius in the spread of antibiotic resistance genes both in the oral sphere and in the gut.     

Complete Genes May Pass from Food to Human Blood

Our bloodstream is considered to be an environment well separated from the outside world and the digestive tract. According to the standard paradigm large macromolecules consumed with food cannot pass directly to the circulatory system. During digestion proteins and DNA are thought to be degraded into small constituents, amino acids and nucleic acids, respectively, and then absorbed by a complex active process and distributed to various parts of the body through the circulation system. Here, based on the analysis of over 1000 human samples from four independent studies, we report evidence that meal-derived DNA fragments which are large enough to carry complete genes can avoid degradation and through an unknown mechanism enter the human circulation system. In one of the blood samples the relative concentration of plant DNA is higher than the human DNA. The plant DNA concentration shows a surprisingly precise log-normal distribution in the plasma samples while non-plasma (cord blood) control sample was found to be free of plant DNA.     

Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species from the Human Gastrointestinal Tract

One hundred and twenty-two strains of Bifidobacterium and Lactobacillus species have been tested against 12 antibiotics and two antibiotic mixtures by a commercial system (Sensititre Anaero3; Treck Diagnostic Systems). The upper limits of some minimum inhibitory concentrations (MICs) were completed on MRS agar plates by the NCCLS procedure. All strains were sensitive to chloramphenicol and imipenem and most of the strains were resistant to metronidazole. Bifidobacteria isolates were susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Approximately 30% of the Bifidobacterium isolates were resistant to tetracycline, as well as five Lactobacillus strains belonging to four different species. None of the tested Bifidobacterium isolates was resistant to vancomycin, whereas a species-dependent resistance was found among the lactobacilli. Single strains of Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus brevis were resistant to erythromycin and/or clindamycin. Most of the observed resistances seemed to be intrinsic, but some others could be compatible with transmissible determinants.     

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10⁻⁶ per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.     

Antibiotic Resistance Patterns of Enterococci and Occurrence of Vancomycin-Resistant Enterococci in Raw Minced Beef and Pork in Germany

The food chain, especially raw minced meat, is thought to be responsible for an increase in the incidence of vancomycin-resistant enterococci (VRE) in human nosocomial infections. Therefore, 555 samples from 115 batches of minced beef and pork from a European Union-licensed meat-processing plant were screened for the occurrence of VRE. The processed meat came from 45 different slaughterhouses in Germany. Enterococci were isolated directly from Enterococcosel selective agar plates and also from Enterococcosel selective agar plates supplemented with 32 mg of vancomycin per liter. In addition, peptone broth was used in a preenrichment procedure, and samples were subsequently plated onto Enterococcosel agar containing vancomycin. To determine resistance, 209 isolates from 275 samples were tested with the glycopeptides vancomycin, teicoplanin, and avoparcin and 19 other antimicrobial substances by using a broth microdilution test. When the direct method was used, VRE were found in 3 of 555 samples (0.5%) at a concentration of 1.0 log CFU/g of minced meat. When the preenrichment procedure was used, 8% of the samples were VRE positive. Our findings indicate that there is a low incidence of VRE in minced meat in Germany. In addition, the resistance patterns of the VRE isolates obtained were different from the resistance patterns of clinical isolates. A connection between the occurrence of VRE in minced meat and nosocomial infections could not be demonstrated on the basis of our findings.     

Marine Sediment Bacteria Harbor Antibiotic Resistance Genes Highly Similar to Those Found in Human Pathogens

The ocean is a natural habitat for antibiotic-producing bacteria, and marine aquaculture introduces antibiotics into the ocean to treat infections and improve aquaculture production. Studies have shown that the ocean is an important reservoir of antibiotic resistance genes. However, there is a lack of understanding and knowledge about the clinical importance of the ocean resistome. We investigated the relationship between the ocean bacterial resistome and pathogenic resistome. We applied high-throughput sequencing and metagenomic analyses to explore the resistance genes in bacterial plasmids from marine sediments. Numerous putative resistance determinants were detected among the resistance genes in the sediment bacteria. We also found that several contigs shared high identity with transposons or plasmids from human pathogens, indicating that the sediment bacteria recently contributed or acquired resistance genes from pathogens. Marine sediment bacteria could play an important role in the global exchange of antibiotic resistance.     

Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

The Incidence of Antibiotic Resistance among Coliform Bacteria Isolated from Food

Three-hundred-and-seventy-eight strains of coliform bacteria were isolated from specimens of commonly sold milk and food products. Klebsiella and Enterobacter spp. were predominating. Resistance to sulphonamides, streptomycin, and chloramphenicol occurred in only 5, 1, and 2 strains, respectively. No tetracycline-resistant strains were found. Two-hundred-and-two strains (54 %) were resistant to ampicillin. In genetic crosses with a sensitive strain of E. coli Κ 12 W 3132 transmissible R factors could not be demonstrated in any of the resistant coliform strains. It is concluded that food is not a significant source of antibiotic resistant enteric bacteria. It may, however, be suggested that food is a source of potentially pathogenic gram-negative bacteria which points out the importance of strict hygienic surveillance of food production.     

狍冬季消化道形态特征及食物颗粒分布

1999年1月在黑龙江省通河县小东林场猎杀14只狍,应用解剖学方法和湿筛法分别对狍冬季消化道形态特征及消化道内食物颗粒分布进行了研究。结果表明,狍复胃中瘤胃比例最大,占71.3±2.6%,其次为皱胃(11.5士2.2%),瓣胃(9.7士2.3%),网胃(7.6士0.9%0);瘤胃粘膜中,瘤胃前庭区(ACF)是最重要的消化和吸收部位之一,具有最大的乳突密度、乳突长度和粘膜赓面扩张系数,显著高于其它区域(P< 0.05 ),后背盲囊区(DBF)次之;网瓣口在狍食物颗粒流通中起到了重要的调节作用,消化道食物颗粒分布中,大颗粒食物(>1.00mm)在瘤网胃中的比例为35.06±10.76%,显著高于其在瓣胃、皱胃及其它消化道内的比例(P<0.05),而小于1.00 mm的食物颗粒在瓣胃以下消化道中有明显的增加(P<0.05), 1.00mm为冬季通过狍网瓣口的临界食物颗粒大小。狍为适应冬季食物营养质量下降,通过增加瘤网胃容量、缩小网瓣口来改变消化道形态而增加食物颗粒在瘤网胃内的滞留时间,使富含纤维素的食物充分发酵和分解以获得更多的能量,从而产生营养适应性的季节变化。     

Dynamics of Meloidogyne incognita Virulence to Resistance Genes Rk and Rk2 in Cowpea

The virulence index of three Meloidogyne incognita field isolates to the resistance gene Rk in cowpea was 0%, 75%, and 120%, with the index measured as reproduction on resistant plants as a percentage of the reproduction on susceptible plants. Continuous culture of the 75% virulent isolate on susceptible tomato for more than 5 years (about 25 generations) resulted in virulence decline to about 4%. The rate of the decline in virulence was described by exponential decay, indicating the progressive loss of virulence on a susceptible host. The 120% virulent isolate declined to 90% virulence during five generations on susceptible cowpea. Following virulence decline, the two isolates were compared over 5 years in inoculated field microplots both separately and as a mixture on susceptible, gene Rk, and gene Rk² cowpea plants. At infestation of the plots, the two isolates were 1.2% and 92.0% virulent, respectively, to gene Rk and 0.2% and 8.1% virulent, respectively, to gene Rk². Virulence to gene Rk in the two isolates and in mixture increased under 5 years of continuous Rk cowpea plants to 129% to 172% and under Rk² cowpea plants to 113% to 139 % by year 5. Virulence to gene Rk² increased during continuous cropping with Rk cowpea plants to 42% to 47% and with Rk² cowpea plants to 22% to 48% by year 5. Selection of Rk²-virulence was slower in the isolate with low itt-virulence. The virulence to both genes Rk and Rk² in the mixed population was not different from that in the highly virulent isolate by year 5 of all cropping combinations. Selection of Rk²-virulence on plants with Rk, and vice versa, indicated at least partial overlap of gene specificity between Rk and Rk² with respect to selection of nematode virulence. This observation should be considered when resistance is used in cowpea rotations.     

In Situ Transfer of Antibiotic Resistance Genes from Transgenic (Transplastomic) Tobacco Plants to Bacteria

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.