Benzalkonium chloride tolerance of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> strains isolated from a meat processing facility is related to presence of plasmid-borne <Emphasis Type="Italic">bcr</Emphasis>ABC cassette

Listeria monocytogenes is a serious foodborne pathogen capable of persisting in food processing environments. Tolerance to disinfectants used in industrial settings constitutes an important factor of Listeria survival. In the present study, the mechanism of tolerance to benzalkonium chloride (BAC) was investigated in 77 L. monocytogenes isolates from a meat facility. By PCR approach, the mdrL and lde chromosomal efflux pump genes were detected in all isolates. No isolate was positive for qacH and emrE genes. However, the bcrABC cassette was present in 17 isolates of serogroup IIa possessing the same AscI/ApaI pulsotype, the operon being localized on a plasmid. The significant relation of BAC tolerance with bcrABC presence was confirmed as all bcrABC positive isolates showed the highest minimal inhibitory concentration (MIC) values for BAC and increased sensitivity to BAC was observed after plasmid curing. No effect of the efflux pump inhibitor reserpine on BAC tolerance in bcrABC positive strains was observed in contrast to all bcrABC negative strains. Lower ethidium bromide efflux in bcrABC positive isolates compared to bcrABC negative and plasmid-cured L. monocytogenes isolates was observed. The expression of bcrABC genes was BAC-induced. The confirmed effect of bcrABC to increased BAC tolerance, coupled with its plasmid location, may be an important factor in potential dissemination of the biocide resistance among Listeria species. The understanding of molecular mechanisms of biocide tolerance should help to improve control measures to prevent further spread of L. monocytogenes in food production environments with frequent use of BAC.     

Genomic characterization of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> isolates reveals that their persistence in a pig slaughterhouse is linked to the presence of benzalkonium chloride resistance genes

Background

The aim of this study was to characterize the genomes of 30?Listeria monocytogenes isolates collected at a pig slaughterhouse to determine the molecular basis for their persistence.

Results

Comparison of the 30?L. monocytogenes genomes showed that successive isolates (i.e., persistent types) recovered from thew sampling site could be linked on the basis of single nucleotide variants confined to prophage regions. In addition, our study revealed the presence among these strains of the bcrABC cassette which is known to produce efflux pump-mediated benzalkonium chloride resistance, and which may account for the persistence of these isolates in the slaughterhouse environment. The presence of the bcrABC cassette was confirmed by WGS and PCR and the resistance phenotype was determined by measuring minimum inhibitory concentrations. Furthermore, the BC-resistant strains were found to produce lower amounts of biofilm in the presence of sublethal concentrations of BC.

Conclusions

High resolution SNP-based typing and determination of the bcrABC cassette may provide a means of distinguishing between resident and sporadic L. monocytogenes isolates, and this in turn will support better management of this pathogen in the food industry.

     

An evaluation of multidrug-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates in urinary tract infections from Aguascalientes,Mexico: cross-sectional study

Background

Uropathogenic Escherichia coli (UPEC) are one of the main bacteria causing urinary tract infections (UTIs). The rates of UPEC with high resistance towards antibiotics and multidrug-resistant bacteria have increased dramatically in recent years and could difficult the treatment.

Methods

The aim of the study was to determine multidrug-resistant bacteria, antibiotic resistance profile, virulence traits, and genetic background of 110 E. coli isolated from community (79 isolates) and hospital-acquired (31 isolates) urinary tract infections. The plasmid-mediated quinolone resistance genes presence was also investigated. A subset of 18 isolates with a quinolone-resistance phenotype was examined for common virulence genes encoded in diarrheagenic and extra-intestinal pathogenic E. coli by a specific E. coli microarray.

Results

Female children were the group most affected by UTIs, which were mainly community-acquired. Resistance to trimethoprim–sulfamethoxazole, ampicillin, and ampicillin–sulbactam was most prevalent. A frequent occurrence of resistance toward ciprofloxacin (47.3%), levofloxacin (43.6%) and cephalosporins (27.6%) was observed. In addition, 63% of the strains were multidrug-resistant (MDR). Almost all the fluoroquinolone (FQ)-resistant strains showed MDR-phenotype. Isolates from male patients were associated to FQ-resistant and MDR-phenotype. Moreover, hospital-acquired infections were correlated to third generation cephalosporin and nitrofurantoin resistance and the presence of kpsMTII gene. Overall, fimH (71.8%) and fyuA (68.2%), had the highest prevalence as virulence genes among isolates. However, the profile of virulence genes displayed a great diversity, which included the presence of genes related to diarrheagenic E. coli. Out of 110 isolates, 25 isolates (22.7%) were positive to qnrA, 23 (20.9%) to qnrB, 7 (6.4%) to qnrS1, 7 (6.4%) to aac(6′)lb-cr, 5 (4.5%) to qnrD, and 1 (0.9%) to qnrC genes. A total of 12.7% of the isolates harbored bla_CTX-M genes, with bla_CTX-M-15 being the most prevalent.

Conclusions

Urinary tract infection due to E. coli may be difficult to treat empirically due to high resistance to commonly used antibiotics. Continuous surveillance of multidrug resistant organisms and patterns of drug resistance are needed in order to prevent treatment failure and reduce selective pressure. These findings may help choosing more suitable treatments of UTI patients in this region of Mexico.

     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

Severity of drug resistance and co-existence of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in diabetic foot ulcer infections

The genes encoding aminoglycoside resistance in Enterococcus faecalis may promote collateral aminoglycoside resistance in polymicrobial wounds. We studied a total of 100 diabetic foot ulcer samples for infection and found 60 samples to be polymicrobial, 5 to be monomicrobial, and 35 samples to be culture negative. A total of 65 E. faecalis isolates were screened for six genes coding for aminoglycoside resistance, antibiotic resistance patterns, and biofilm production. Infectious Diseases Society of America/International Working Group on the Diabetic Foot system was used to classify the wound ulcers. Majority of the subjects with culture-positive wound were recommended conservative management, while 14 subjects underwent amputation. Enterococcal isolates showed higher resistance for erythromycin, tetracycline, and ciprofloxacin. Isolates from grade 3 ulcer showed higher frequency of aac(6′)-Ie-aph(2″)-Ia, while all the isolates were negative for aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id. The isolates from grade 3 ulcers showed higher resistance to aminoglycosides as well as teicoplanin and chloramphenicol. All the 39 biofilm producers were obtained from polymicrobial wound and showed higher resistance when compared to biofilm non-producers. Higher frequency of isolates carrying aac(6′)-Ie-aph(2″)-Ia in polymicrobial community showing resistance to key antibiotics suggests widespread distribution of aminoglycoside-resistant E. faecalis and their role in worsening diabetic foot ulcers.     

Genotyping of Antimicrobial Resistance and Virulence in <Emphasis Type="Italic">Staphylococcus</Emphasis> Isolated from Food of Animal Origin in Mexico

Ninety-six methicillin-susceptible Staphylococcus aureus (MSSA) and 11 methicillin-resistant coagulase-negative staphylococci (MRCNS) were recovered from food of animal origin. Multi-drug resistance was detected in 34.1% of isolates. Tetracycline-resistant staphylococci harbored tetK gene (68.8%). Erythromycin/clindamycin-resistant staphylococci carried lnuA/lnuB genes frequently alone or combined with msrA gene. The sec gene was detected in 15.6% of MSSA and two isolates harbored the immune evasion cluster. The spa t337 predominated among MSSA strains. Two ermC-positive MRCNS isolates were observed, five mecA-positive carried SCCmec IVa and 6 were non-typeable by the IWG-SCC classification. These results demonstrate that food of animal origin can be a potential source for spreading of multidrug-resistance gene.     

Epidemiological relationships of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> strains isolated from humans and chickens in South Korea

Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007–2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009–2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.     

Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3′)-Ia and aac(6′)-aph(2″) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans.     

Diversity of species and antibiotic resistance in enterococci isolated from seafood in Tunisia

The purpose of this study was to analyse the antibiotic resistance and virulence of enterococci recovered from seafood and to characterise the associated genes. Forty-four enterococcal isolates [Enterococcus faecalis (21), E. faecium (11), E. casseliflavus (5), E. durans (3), E. hirae (2), E. gallinarum (1) and E. mundtii (1)] were recovered from 70 samples of seafood collected during March–May 2015 in Tunisia. Isolates were tested for antibiotic resistance to 12 antibiotics by the disc diffusion method. Rates of resistance in the range 25–45.5% were observed for pristinamycin, ciprofloxacin, streptomycin, tetracycline and erythromycin, and in the range 6.8–9.1% for kanamycin, gentamicin and chloramphenicol. However, all strains showed susceptibility to β-lactams and glycopeptides. Multi-resistance to at least three different classes of antibiotics was detected in 14 strains (31.8%). Among 12 tetracycline-resistant enterococci, tet(M) was detected in 11 isolates and tet(L) in seven isolates. The erm(B) gene was identified in 91% of erythromycin-resistant isolates. All chloramphenicol-resistant isolates carried the cat gene, and all kanamycin-resistant isolates harboured the aph(3)-IIIa gene. The aac(6′)-aph(2″) and ant(6)-Ia genes were detected in high-level gentamicin- and streptomycin-resistant isolates, respectively. The virulence genes gelE (29.5%), esp (9.1%), cylA and cylB (9.1%) were found in enterococci. This is the first study in Tunisia to underscore the importance of seafood as a reservoir of enterococci carrying resistance and virulence genes.     

Genomic characterisation,detection of genes encoding virulence factors and evaluation of antibiotic resistance of <Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from cattle with clinical metritis

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.     

Expression Patterns of ABC Transporter Genes in Fluconazole-Resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

Clinical management of fungal diseases is compromised by the emergence of antifungal drug resistance in fungi, which leads to elimination of available drug classes as treatment options. An understanding of antifungal resistance at molecular level is, therefore, essential for the development of strategies to combat the resistance. This study presents the assessment of molecular mechanisms associated with fluconazole resistance in clinical Candida glabrata isolates originated from Iran. Taking seven distinct fluconazole-resistant C. glabrata isolates, real-time PCRs were performed to evaluate the alternations in the regulation of the genes involved in drug efflux including CgCDR1, CgCDR2, CgSNQ2, and CgERG11. Gain-of-function (GOF) mutations in CgPDR1 alleles were determined by DNA sequencing. Cross-resistance to fluconazole, itraconazole, and voriconazole was observed in 2.5 % of the isolates. In the present study, six amino acid substitutions were identified in CgPdr1, among which W297R, T588A, and F575L were previously reported, whereas D243N, H576Y, and P915R are novel. CgCDR1 overexpression was observed in 57.1 % of resistant isolates. However, CgCDR2 was not co-expressed with CgCDR1. CgSNQ2 was upregulated in 71.4 % of the cases. CgERG11 overexpression does not seem to be associated with azole resistance, except for isolates that exhibited azole cross-resistance. The pattern of efflux pump gene upregulation was associated with GOF mutations observed in CgPDR1. These results showed that drug efflux mediated by adenosine-5-triphosphate (ATP)-binding cassette transporters, especially CgSNQ2 and CgCDR1, is the predominant mechanism of fluconazole resistance in Iranian isolates of C. glabrata. Since some novel GOF mutations were found here, this study also calls for research aimed at investigating other new GOF mutations to reveal the comprehensive understanding about efflux-mediated resistance to azole antifungal agents.     

Beneficial and Safety Properties of a <Emphasis Type="Italic">Corynebacterium vitaeruminis</Emphasis> Strain Isolated from the Cow Rumen

Corynebacterium vitaeruminis MRU4 was isolated from the cow rumen and was differentiated from other isolates by rep-PCR and RAPD and identified by 16S rRNA sequencing. This strain presented higher survival rates for low pH and bile salts treatments, and it was able to survive and multiply in simulated gastric and intestinal environments. C. vitaeruminis MRU4 had a 53.2% auto-aggregation rate, 42.4% co-aggregation rate with Listeria monocytogenes Scott A, 41.6% co-aggregation rate with Enterococcus faecalis ATCC 19443, 10.0% co-aggregation rate with Lactobacillus sakei ATCC 15521, and 98.2% cell surface hydrophobicity rate. PCR analysis showed the presence of EFTu and map genes. The strain possessed positive results for deconjugation of bile salts (taurocholic acid, taurodeoxycholic acid, glycocholic acid, and glycodeoxycholic acid) and positive results for β-galactosidase activity and lactose assimilation activity (glucose of 8.15 ± 0.01 CFU/ml and lactose of 9.24 ± 0.02 CFU/ml). No virulence was observed by phenotypical tests. C. vitaeruminis MRU4 was resistant to oxacillin, gentamicin, erythromycin, clindamycin, sulfa/trimethoprim, and rifampicin by the disc diffusion method and showed resistance just for vancomycin by the Etest® strips test. The strain was negative for 50 tested virulence and resistance genes based on performed PCR. Based on our knowledge, this is the first report regarding the beneficial potential of one C. vitaeruminis strain.     

Colistin resistance in <Emphasis Type="Italic">Enterobacter</Emphasis> spp. isolates in Korea

We investigated the colistin resistance rate among 356 Enterobacter spp. clinical isolates from eight hospitals in Korea. Antibiotic susceptibility testing was performed by broth microdilution. While 51 of 213 (23.9%) Enterobacter cloacae isolates were colistin-resistant, only six of 143 (4.2%) E. aerogenes isolates showed resistance. We also identified the skip well phenotype in eight E. cloacae and three E. aerogenes isolates. Multilocus sequence typing for E. cloacae and randomly amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus PCR for E. aerogenes revealed that clonal spreading of colistin-resistant and skip well Enterobacter spp. isolates had not occurred. In vitro time-kill assays were performed with three colistin-resistant, three skip well, and two colistin-susceptible isolates of E. cloacae and E. aerogenes. Inconsistent results were observed among isolates with skip well phenotypes; while some were eradicated by 2 mg/L colistin, others were not. This suggests that skip well isolates have differentiated into different categories. As the high rates of colistin resistance in E. cloacae detected are of clinical concern, continuous monitoring is warranted. In addition, the clinical implications and mechanisms of the skip well phenotype should be investigated to ensure the appropriate use of colistin against Enterobacter infections.     

Improvement of seedling and panicle blast resistance in <Emphasis Type="Italic">Xian</Emphasis> rice varieties following <Emphasis Type="Italic">Pish</Emphasis> introgression

Rice blast is a serious disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae. Incorporating disease resistance genes in rice varieties and characterizing the distribution of M. oryzae isolates form the foundation for enhancing rice blast resistance. In this study, the blast resistance gene Pish was observed to be differentially distributed in the genomes of rice sub-species. Specifically, Pish was present in 80.5% of Geng varieties, but in only 2.3% of Xian varieties. Moreover, Pish conferred resistance against only 23.5% of the M. oryzae isolates from the Geng-planting regions, but against up to 63.2% of the isolates from the Xian-planting regions. Thus, Pish may be an elite resistance gene for improving rice blast resistance in Xian varieties. Therefore, near-isogenic lines (NILs) with Pish and the polygene pyramid lines (PPLs) PPL^Pish/Pi1, PPL^Pish/Pi54, and PPL^Pish/Pi33 in the Xian background Yangdao 6 were generated using a molecular marker-assisted selection method. The results suggested that (1) Pish significantly improved rice blast resistance in Xian varieties, which exhibited considerably improved seedling and panicle blast resistance after Pish was introduced; (2) PPLs with Pish were more effective than the NILs with Pish regarding seedling and panicle blast resistance; (3) the PPL seedling and panicle blast resistance was improved by the complementary and overlapping effects of different resistance genes; and (4) the stability of NIL and PPL resistance varied under different environmental conditions, with only PPL^Pish/Pi54 exhibiting highly stable resistance in three natural disease nurseries (Jianyang, Jinggangshan, and Huangshan). This study provides new blast resistance germplasm resources and describes a novel molecular strategy for enhancing rice blast resistance.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> isolated from cheese: production and partial characterization of bacteriocin B391

Lactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.     

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> isolated from raw milk and unpasteurized traditional cheeses in Romania

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla _SHV , bla _CMY , bla _TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n = 13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n = 8). A high percent (33.3%; n = 9) revealed the beta-lactamase (bla _TEM ) resistance gene, and none of the samples tested positive for bla _CMY and bla _SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n = 4) and 7.4% (n = 2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.     

Assessment of three indigenous South African herbivores as potential reservoirs and vectors of antibiotic-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Due to limited data available on the presence of antibiotic-resistant (ABR) bacteria in faeces of wild herbivores in South Africa, this study analysed resistance patterns for Escherichia coli isolates from wildebeest, zebra and giraffe in addition to pet and farm pig faeces. Total and faecal coliforms and E. coli were quantified in faecal matter using a most probable number (MPN) guideline procedure. Antibiotic resistance profiles against 12 selected antibiotics representing seven classes were determined for 30 randomly selected E. coli isolates from each animal using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion procedure. While log₁₀ MPN values per gram of animal faeces for total/faecal coliforms ranged from 4.51/4.11 to 5.70/5.50, the E. coli MPN values were in a range of 3.43–5.14. The proportion of ABR E. coli isolates ranged from 43% (giraffe) to 93% (zebra). About 47% of E. coli isolates from zebra faeces were categorized as multidrug-resistant (MDR), while for wildebeest and giraffe, no MDR isolates were detected. In comparison, 10% of E. coli isolates from pet pig and about 7% from farm pig faeces were categorized as MDR. Although most MDR isolates were resistant to at least one β-lactam antibiotic, only one MDR isolate from farm pig faeces was resistant to both norfloxacin and ciprofloxacin, the two fluoroquinolones tested. However, no resistance was detected to the tested carbapenems and tigecycline. The results of this study indicate that indigenous South African herbivores may serve as potential reservoirs and vectors for the dissemination of ABR E. coli strains.