Detection of Extended‐Spectrum β‐Lactamase and Plasmid‐Mediated Quinolone Resistance Determinants in Escherichia coli Isolates from Retail Meat in China

The aim of this study was to investigate the presence of extended‐spectrum β‐lactamase (ESBL) and plasmid‐mediated quinolone resistance (PMQR) genes in Escherichia coli isolated from retail meat samples in Henan Province, China. E. coli isolates were detected in 179 of 645 (27.7%) retail meat samples. Resistance of these isolates to antimicrobials was commonly observed, with 78.2% of isolates resistant to streptomycin, 74.3% resistant to tetracycline and 54.2% resistant to trimethoprim/sulfamethoxazole. Of the 179 isolates, 30 (16.7%) expressed ESBL, with bla_TEM‐1 (n = 17) and bla_CTX‐M‐14 (n = 9) most commonly mediating the ESBL phenotype. PMQR genes were present in 14 isolates (7.8%), with qnr and aac(6′)‐Ib‐cr detected alone or in combination in nine (5.0%) and seven isolates (3.9%), respectively. The qnr genes detected included qnrS1 (n = 5), qnrA1 (n = 3), and qnrB4 (n = 1). The qepA gene was absent among these isolates. CTX‐M‐14 was the most prevalent ESBL type among the PMQR‐positive isolates. The qnr and aac(6′)‐Ib‐cr genes were found to co‐reside and be co‐transferred with bla_CTX‐M‐14 or bla_TEM‐1 in five isolates. Our data suggest that retail meat may act as a reservoir for multi‐resistant E. coli and may facilitate the dissemination of resistance genes.     

Short communication: Extended-spectrum cephalosporin-resistant Escherichia coli in colostrum from New Brunswick,Canada, dairy cows harbor blaCMY-2 and blaTEM resistance genes

Dairy calves are colonized shortly after birth by multidrug resistant (MDR) bacteria, including Escherichia coli. The role of dairy colostrum fed to calves as a potential source of MDR bacteria resistance genes has not been investigated. This study determined the recovery rate of extended-spectrum cephalosporin-resistant (ESC-R) E. coli in colostrum from cows. The ESC-R E. coli isolates were further investigated to determine their phenotypic antimicrobial resistance pattern and the genes conferring ESC-R. Fresh colostrum was collected from 452 cows from 8 dairy herds in New Brunswick, Canada. The ESC-R E. coli was isolated from the colostrum by using the VACC agar, a selective media for extended-spectrum β-lactamase producing Enterobacteriaceae. Minimum inhibitory concentration was determined for all the suspected ESC-R E. coli isolates using a commercial gram-negative broth microdilution method. Two multiplex PCR were conducted on all the suspected ESC-R E. coli isolates to determine the presence of the bla_CTX-M (groups 1, 2, 9, and 8/25) bla_CMY-2, bla_SHV, and bla_TEM resistance genes. The ESC-R E. coli were detected in 20 (4.43%) of the colostrum samples. At least 1 ESC-R E. coli isolate was detected in 6 (75%) of the dairy herds. All ESC-R E. coli had MDR profiles based on minimum inhibitory concentration testing. No bla_CTX-M groups genes were detected; however, the bla_CMY-2 gene was detected in 9 or 20 (45%) and bla_TEM was detected in 7 of 20 (35%) of the ESC-R E. coli. No ESC-R E. coli had both bla_CMY-2 and bla_TEM resistance genes. This is the first report of bla_CMY-2 and bla_TEM genes found in E. coli isolates cultured from dairy colostrum to our knowledge.     

Antimicrobial susceptibility and characterization of extended-spectrum β-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015

In this study, we aimed to assess trends in antimicrobial resistance and to investigate the characteristics of extended-spectrum β-lactamase (ESBL)-producing isolates from bovine mastitic milk from 2012 to 2015. A total of 374 Escherichia coli isolates were analyzed (154 in 2012, 113 in 2013, 76 in 2014, and 31 in 2015). No consistent trends in antimicrobial resistance of E. coli isolates occurred during the 4-yr period. The most frequently observed resistance was tetracycline (23.3%), followed by streptomycin (17.1%), ampicillin (16.6%), neomycin (11.8%), and trimethoprim/sulfamethoxazole (11.2%). Multidrug resistance was observed in 15.5% of isolates. Among these isolates, 15 (4.0%) carried one or more bla_CTX-M and AmpC ESBL genes from 11 different farms, including bla_CTX-M-15 at 4 farms, bla_CTX-M-3 at 2 farms, bla_CTX-M-1 at 3 farms, and bla_CMY-2 at 3 farms. This study is the first report of bla_CTX-M-3-producing E. coli in dairy milk. Transfer of ESBL was observed in 3 bla_CTX-M-3-producing isolates, 1 bla_CTX-M-1-producing isolate, and all 3 bla_CMY-2-producing isolates. Almost all bla_CTX-M-15 and bla_CTX-M-1 genes possessed an insertion sequence, ISECP1, upstream of the bla_CTX-M gene. Identical pulsed-field gel electrophoresis profiles were also observed in bla_CTX-M-producing E. coli from the same farm. These results suggested that ESBL might spread by both clonal and horizontal spread in dairy farms in South Korea. Although no significant changes occurred in the antimicrobial resistance of E. coli during the 4-yr study period, the resistance rates and presence of ESBL were high compared with those in other countries. Thus, these findings suggest the importance of control measures for E. coli, particularly ESBL-producing bacteria, on dairy farms to reduce treatment failure and transmission to humans.     

Characterization of mammary pathogenic Escherichia coli reveals the diversity of Escherichia coli isolates associated with bovine clinical mastitis in Brazil

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN⁺) and lack the bundle-forming pilus (bfpB^?) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum β-lactamase–producing E. coli were identified, both harboring bla_CTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor–encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum β-lactamase–producing isolates can turn this veterinary medicine problem into a public health concern.     

Characteristics of Escherichia coli from raw vegetables at a retail market in the Czech Republic

A large epidemic caused by shigatoxigenic Escherichia coli (E. coli) in spring 2011 in Germany resulted in reduction of trust in the health safety of raw vegetables and sprouted seeds. This study focused on the detection and characterization of E. coli in raw vegetables and sprouted seeds sold in the Czech Republic. Out of 91 samples, 24 (26.4%) were positive for the presence of E. coli. Resistance to antimicrobial agents was determined by the disk diffusion method and E-test. Polymerase chain reaction was used for the detection of selected genes encoding virulence — eaeA, hly, stx₁, and stx₂ and genes encoding resistance to tetracycline — tet(A), tet(B), tet(C), and tet(G) and to β-lactams — bla_TEM, bla_SHV, and bla_CTX. The bla_TEM gene was detected in two isolates, the tet(B) gene in three and tet(A) in one isolate. No hly, stx₁, or stx₂ genes were present, but the eaeA gene was found in three (11.1%) isolates from imported vegetables. These isolates can be considered as potentially enteropathogenic. Results of this study show that raw vegetables and sprouted seeds sold in the retail market can represent a potential risk for consumers.     

Aeromonas spp. simultaneously harbouring blaCTX-M-15, blaSHV-12, blaPER-1 and blaFOX-2, in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea,Croatia

Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired β-lactamases, including extended-spectrum-β-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel (Mytilus galloprovincialis) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and β-lactam/β-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC (bla_FOX, bla_CMY, bla_MOX, bla_LAT, bla_BIL, bla_DHA, bla_ACC, bla_MIR, bla_ACT), ESBLs (bla_TEM, bla_SHV, bla_CTX-M, bla_PER, bla_VEB, bla_GES/IBC, bla_OXA) and integrases (intI1, intI2, intI3). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila) isolates, with bla_CTX-M-15 gene identified in 19 and bla_SHV-12 in 12 isolates. Among them, 10 isolates simultaneously harboured bla_CTX-M-15 and bla_SHV-12, while 3 isolates additionally carried an AmpC β-lactamase bla_FOX-2 gene. bla_PER-1 gene was identified in a single isolate also harbouring the bla_CTX-M-15 gene. While bla_SHV-12 was chromosomally encoded, bla_CTX-M-15 was located on conjugative IncFIB-type plasmids of ~ 40 kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates.     

Isolation and Molecular Characterization of Multidrug-Resistant Strains of Escherichia coli and Salmonella from Retail Chicken Meat in Japan

ABSTRACT: Sixty‐nine Escherichia coli and 10 Salmonella isolates, recovered from retail chicken meat in Hiroshima prefecture, Japan, were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes. Twenty‐eight out of 69 (40.6%) of E. coli and all 10 Salmonella isolates were exhibited multidrug resistance phenotypes. The most commonly reported resistance phenotypes were against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, and trimethoprim/sulfamethoxazole. PCR screening for integrons showed that 8 (11.6%) of the E. coli isolates were positive for the class 1 integrons and 1 isolate (1.4%) was positive for the class 2 integrons. Among the 10 Salmonella isolates, 9 were positive for class 1 integrons and none was positive for class 2 integrons. The identified antibiotic resistance gene cassettes within the class 1 integrons were dfrA1, dfrA7, aadA1, aadB, and catB3, while dfrA1, sat2, and aadA1 were identified within class 2 integron. The β‐lactamase resistance gene bla_TEM‐1 was identified in 12 (17.3%) of E. coli isolates and in only one of the Salmonella isolates. The bla_CMY‐2 gene, encoding AmpC β‐lactamase, was detected in 16 (23.2%) of the E. coli isolates only. Conjugation experiments demonstrated that there was plasmid‐mediated transfer of bla_CMY‐2 and bla_TEM‐1. These results highlighted the role of retail chicken meat as a potential source for multidrug‐resistant strains of E. coli and Salmonella. To the best of our knowledge, this is the 1st report of isolation and molecular characterization of multidrug‐resistant strains of E. coli from retail chicken meat in Japan.     

Molecular characterization of antimicrobial resistance in Klebsiella pneumoniae isolated from Brazilian dairy herds

In this observational study, phenotypic and genotypic patterns of antimicrobial resistance (AMR) in Klebsiella pneumoniae isolated from intramammary infections, clinical mastitis, fresh feces, rectal swabs, animal hindlimbs, and bulk tank milk samples from Brazilian dairy herds were investigated. In addition, we identified specific genetic variants present among extended-spectrum β-lactamase (ESBL) producers. We obtained 169 isolates of K. pneumoniae from 2009 to 2011 on 24 Brazilian dairy farms located in 4 Brazilian states. The AMR profile of all isolates was determined using disk-diffusion assays. The antimicrobial panel included drugs commonly used as mastitis treatment in Brazilian dairy herds (gentamicin, cephalosporins, sulfamethoxazole-trimethoprim, tetracycline) as well as antimicrobials of critical importance for human health (meropenem, ceftazidime, fluoroquinolones). The K. pneumoniae isolates resistant to tetracycline, fluoroquinolones, sulfamethoxazole-trimethoprim, or chloramphenicol were screened for presence of drug-specific AMR genes [tet, qnr, aac(6')-Ib, floR, catA2, cm1A, dfr, sul] using PCR. In addition, we identified ESBL genes present among ESBL-producers by using whole genome sequencing. Genomes were assembled and annotated, and patterns of AMR genes were investigated. Resistance was commonly detected against tetracycline (22.5% of all isolates), streptomycin (20.7%), and sulfamethoxazole-trimethoprim (9.5%). Antimicrobial resistance rates were higher in K. pneumoniae isolated from intramammary infections in comparison with isolates from feces (19.2 and 0% of multidrug resistance in intramammary and fecal isolates, respectively). In contrast, no difference in AMR rates was observed when contrasting hind limbs and isolates from intramammary infections. The genes tetA, sul2, and floR were the most frequently observed AMR genes in K. pneumoniae resistant to tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol, respectively. The tetA gene was present exclusively in isolates from milk. The genes bla_CTX-M8 and bla_SHV-108 were present in 3 ESBL-producing K. pneumoniae, including an isolate from bulk tank milk. The 3 isolates were of sequence type 281 and had similar mobile genetic elements and virulence genes. Our study reinforced the epidemiological importance and dissemination of bla_CTX-M-8 pST114 plasmid in food-producing animals in Brazil.     

Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal β-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and β-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.     

The phylogenetic group,antimicrobial susceptibility,and virulence genes of Escherichia coli from clinical bovine mastitis

Bovine mastitis is still a central problem on dairy farms despite control programs, and Escherichia coli is a crucial pathogen during the development of bovine mastitis. The virulence genes, antimicrobial susceptibility, and mortality of mice infected with different E. coli isolates from bovine mastitis were determined in this study. According to the presence of the specific genes chuA, yjaA, and TspE4.C2, these isolates mainly belonged to 2 different groups: group A (47/79) and group B1 (22/79). The ompC gene was detected in all the isolates, followed by fimH (89.9%), ECs3703 (88.6%), and ompF (73.4%), whereas most of the virulence genes were not detected in these isolates. The results of the antimicrobial susceptibility tests indicated that the isolates were susceptible to the fluoroquinolones and aminoglycosides. An inverse relationship was shown between the expression level of ompF and antimicrobial resistance; additionally, the isolates that were nonsusceptible to at least 4 classes of antimicrobial agents showed a lower mortality to mice in comparison with the susceptible isolates. This study indicated that antibiotic resistance had emerged in E. coli from bovine mastitis in this area, and appropriate measures should be taken to avoid potential threats to humans and other animals.