Chloramphenicol-resistant Isolates Research Articles

Development of pBACpAK entrapment vector derivatives to detect intracellular transfer of mobile genetic elements within chloramphenicol resistant bacterial isolates

Antimicrobial resistance disseminates throughout bacterial populations via horizontal gene transfer, driven mainly by mobile genetic elements (MGEs). Entrapment vectors are key tools in determining MGE movement within a bacterial cell between different replicons or between sites within the same replicon. The pBACpAK entrapment vector has been previously used to study intracellular transfer in Gram-negative bacteria however since pBACpAK contains a chloramphenicol resistance gene, it cannot be used in bacterial isolates which are already resistant to chloramphenicol. Therefore, we developed new derivatives of the pBACpAK entrapment vector to determine intracellular transfer of MGEs in an Escherichia coli DH5α transconjugant containing the chloramphenicol resistance plasmid pD25466. The catA1 of pBACpAK was replaced by both mcr-1 in pBACpAK-COL and aph(3′)-Ia in pBACpAK-KAN, allowing it to be used in chloramphenicol resistant strains. The plasmid constructs were verified and then used to transform the E. coli DH5α/pD25466 transconjugants in order to detect intracellular movement of the MGEs associated with the pD25466 plasmid. Here we report on the validation of the expanded suite of pBACpAK vectors which can be used to study the intracellular transfer of MGEs between, and within, replicons in bacteria with different antimicrobial resistance profiles.

Resurgence of Chloramphenicol Resistance in Methicillin-Resistant Staphylococcus aureus Due to the Acquisition of a Variant Florfenicol Exporter (fexAv)-Mediated Chloramphenicol Resistance in Kuwait Hospitals.

Following a surge in the prevalence of chloramphenicol-resistant methicillin-resistant Staphylococcus aureus (MRSA) in Kuwait hospitals, this study investigated the genotypes and antibiotic resistance of the chloramphenicol-resistant isolates to ascertain whether they represented new or a resurgence of sporadic endemic clones. Fifty-four chloramphenicol-resistant MRSA isolates obtained in 2014–2015 were investigated. Antibiotic resistance was tested by disk diffusion and MIC determination. Molecular typing was performed using spa typing, multilocus sequence typing, and DNA microarray. Curing and transfer experiments were used to determine the genetic location of resistance determinants. All 54 isolates were resistant to chloramphenicol (MIC: 32–56 mg/L) but susceptible to florfenicol. Two chloramphenicol-resistance determinants, florfenicol exporter (fexA) and chloramphenicol acetyl transferase (cat), were detected. The fexA-positive isolates belonged to CC5-ST627-VI-t688/t450/t954 (n = 45), CC5-ST5-V-t688 (n = 6), whereas the cat-positives isolates were CC8-ST239-III-t037/t860 (n = 3). While cat was carried on 3.5–4.4 kb plasmids, the location of fexA could not be established. DNA sequencing of fexA revealed 100% sequence similarity to a previously reported fexA variant that confers chloramphenicol but not florfenicol resistance. The resurgence of chloramphenicol resistance was due to the introduction and spread of closely related fexA-positive CC5-ST5-V and CC5-ST627-VI clones.

Comparative Analysis of Chloramphenicol-Resistant Enterococcus faecalis Isolated from Dairy Companies in Korea.

Although chloramphenicol is currently banned from use in livestock, other phenicols, such as florfenicol and thiamphenicol, have been used for the treatment of bacterial infections in domestic cattle in Korea. This study compares the characteristics of chloramphenicol-resistant Enterococcus faecalis isolated from the bulk tank milk of four major dairy companies in Korea. Although the distribution of multidrug resistance patterns showed no significant differences between the four companies, 85 chloramphenicol-resistant Enterococcus faecalis isolates showed a significantly high number of resistances against five or six antimicrobial classes (37.6%, respectively) (p < 0.05). When analyzing the distribution of phenicol resistance genes, 31 (36.5%) isolates only carried the catA gene, and two (6.3%) isolates from company A only carried the cfr gene. No isolates carried the catB or fexA genes. Regarding the distribution of other resistance genes, both the tetL and tetM (45.9%), ermB (82.4%), and both aac(6″)-Ie-aph(2″)-la and ant(6′)-Ia genes (30.6%) showed a high prevalence, and the optrA and poxtA genes were observed separately, each in only two (2.4%) isolates. Our results confirm that the dissemination of chloramphenicol-resistant Enterococcus faecalis and some antimicrobial resistance genes show significant differences between dairy companies. Therefore, our results support that each dairy company should undertake effective surveillance programs to better understand and minimize the emergence of resistance on a multidisciplinary level.

Molecular characteristics of fluoroquinolone-resistant avian pathogenic Escherichia coli isolated from broiler chickens

Avian pathogenic Escherichia coli (APEC) is a major pathogen in the poultry industry worldwide including Korea. In this study, the phenotypic and genotypic characteristics of 33 fluoroquinolone (FQ)-resistant APEC isolates from broilers were analyzed. All FQ-resistant APEC isolates showed amino acid exchanges at both gyrA and parC and high minimal inhibitory concentrations for FQs. A total of 11 (33.3%) isolates were positive for the plasmid-mediated quinolone resistance (PMQR) genes, qnrA (8 isolates) and qnrS (3 isolates), and showed multidrug resistance. Among the 11 PMQR-positive isolates, 1 and 2 isolates carried blaCTX-1 and blaCTX-15, respectively, as extended-spectrum β-lactamase (ESBL) producers, and the non-ESBL gene, blaTEM-1, was found in 4 isolates. Among 3 aminoglycoside-resistant isolates, aac(3)-II was only detected in 1 isolate. All 8 APEC isolates with resistance to tetracycline carried the tetA gene. Overall, 6 of the 7 trimethoprim-sulfamethoxazole-resistant isolates carried the sul1 or sul2 genes, while only 2 of the 8 chloramphenicol-resistant isolates carried the catA1 gene. Although 9 isolates carried class I integrons, only 4 isolates carried the gene cassettes dfrA12-aadA2 (2 isolates), dfrA17-aadA5 (1 isolate), extX-psp-aadA2 (1 isolate), and dfrA27 (1 isolate). The most common plasmid replicon was FIB (8 isolates, 72.7%), followed by K/B (4 isolates, 36.4%). Antimicrobial resistance monitoring and molecular analysis of APEC should be performed continuously to surveil the transmission between poultry farms.

The spread of chloramphenicol-resistant Neisseria meningitidis in Southeast Asia

ObjectivesInvasive disease caused by Neisseria meningitidis is a significant health concern globally, but our knowledge of the prevailing serogroups, antimicrobial susceptibility patterns, and genetics of N. meningitidis in Southeast Asia is limited. Chloramphenicol resistance in N. meningitidis has rarely been reported, but was first described in isolates from Vietnam in 1998. We aimed to characterise eight chloramphenicol resistant meningococcal isolates collected between 2007 and 2018 from diagnostic microbiology laboratories in Cambodia, Thailand and the Lao People's Democratic Republic (Laos). MethodsWhole-genome sequencing was used to generate genome sequences from 18 meningococcal isolates including the eight chloramphenicol resistant isolates. We identified antimicrobial resistance genes present in these strains, and examined the phylogenetic relationships between strains. ResultsThe eight resistant strains all contain the same chloramphenicol resistance gene first described in 1998, and are closely related to each other. Strains resistant to penicillin, tetracycline, and ciprofloxacin were also observed, including a chloramphenicol-resistant strain which has acquired penicillin and ciprofloxacin resistance. ConclusionsThis study suggests that chloramphenicol-resistant N. meningitidis is more widespread than previously thought, and that the previously-identified resistant lineage is now found in multiple countries in Southeast Asia.

Molecular characteristics of antimicrobial resistance determinants and integrons in Salmonella isolated from chicken meat in Korea

Summary The increase and spread of antimicrobial resistance, particularly multidrug resistance (MDR), in Salmonella isolated from food animal pose a serious public health concern worldwide. Therefore, the objective of this study was to characterize antimicrobial resistance and analyze the transferability of integrons and antimicrobial resistance genes in MDR Salmonella isolates from retail chicken meat in Korea. Among the 57 Salmonella isolates, 43 (75.4%) showed resistance to at least 1 antimicrobial agent and 29 (50.9%) exhibited MDR. The highest resistance was to nalidixic acid (73.7%), ampicillin (50.9%), tetracycline (42.1%), chloramphenicol (33.3%), and trimethoprim/sulfamethoxazole (33.3%). β-lactamase–encoding genes were detected in 9 (31.0%) of 29 β-lactam–resistant isolates. These genes were as follows: blaCMY-2 (1 S. Albany), blaCTX-M-79 (1 S. Virchow), and blaCTX-M-15 (6 S. Virchow and 1 S. Enteritidis). Seventeen (70.8%) of the 24 tetracycline-resistant isolates harbored only tetA. Among the 19 trimethoprim/sulfamethoxazole-resistant isolates, sul1 was only identified in 3 (68.4%) isolates. cmlA was detected in 4 (21.1%) of the 19 chloramphenicol-resistant isolates. Both aminoglycoside-resistant isolates were positive for ant(2″)-I. Class 1 integrons were observed in 28 (49.1%) isolates belonging to S. Albany (21 isolates), S. Virchow (6 isolate), and S. Enteritidis (1 isolate). All 29 MDR isolates carried one or more integrons and antimicrobial resistance genes. All isolates harboring the ESBL and pAmpC genes exhibited resistance to at least 4 different classes of antimicrobials, including cephalosporins and noncephalosporins. Furthermore, most (79.3%) of the MDR isolates carried 1 or 2 of conjugable plasmids, and the transfer of integrons and resistance genes via plasmids was confirmed in transconjugants, which showed resistance patterns similar to those of the donor strains.

Genetic characterization of high-level aminoglycoside-resistant Enterococcus faecalis and Enterococcus faecium isolated from retail chicken meat

Retail chicken meat can play a role in the transfer of drug resistance to humans through the handling or ingestion of improperly cooked meat contaminated with resistant enterococci. In fact, high-level aminoglycoside-resistance (HLAR) in enterococci identified in human cases. Therefore, the prevalence and genetic characterization of HLAR in enterococci in retail chicken meat were investigated in this study. Of the 345 enterococci strains, 29 (8.7%) showed HLAR. All HLAR in enterococci carried at least 1 of 2 aminoglycoside-modifying enzyme genes, aac(6')Ie-aph(2″)-Ia and ant(6)-Ia. Among the 13 isolates that carried aac(6')Ie-aph(2″)-Ia, 3 had pattern A, with IS256 at both ends, and the other 10 had pattern D, without IS256 at both ends. All HLAR in enterococci also showed multidrug resistance. Among the 24 erythromycin-resistant enterococci, 19 (79.2%) harbored the ermB gene, and one (4.2%) harbored both the ermB and ermA genes. A total of 21 enterococci were tetracycline-resistant and harbored one or more of the following tetracycline resistance genes tet(M), tet(L), and tet(O). The Int-Tn gene was detected in one isolate (3.4%) carrying the tet(M) and ermB genes. All 4 chloramphenicol-resistant isolates carried either the phenicol resistance gene cfr alone (one isolate), both cfr and fexA (one isolate), or both fexA and optrA (2 isolates). Four efflux pump genes, efr(A), efr(B), emeA, and lsa, were detected in all HLAR in Enterococcus faecalis isolates. These results improve our understanding of the transmission dynamics of HLAR in enterococci from non-hospital sources to humans.

Antimicrobial Resistance and Genetic Diversity of Salmonella Serotypes Recovered from Edible Pork Offal from Korea.

Although edible offal can be easily contaminated with foodborne bacteria and regarded as important transfers of antimicrobial resistance to humans, the characterization of bacteria from edible offal have not been researched sufficiently. This study is the first to focus on the molecular characterization of Salmonella isolated from edible pork offal. From a total of 52 Salmonella isolates from edible pork offal, 44 (80.7%) were resistant to at least one antimicrobial agent and 24 (46.2%) exhibited multidrug resistance (MDR). All MDR Salmonella were also resistant to β-lactams and 12 (50.0%) of the isolates were positive for blaTEM-1. Eleven (68.8%) of the 16 gentamicin-resistant isolates harbored the ant(2'')-I gene. Among 18 tetracycline-resistant isolates, tetA and tetB genes were found in 9 (50.0%) and 3 (16.7%) isolates, respectively. The sul1 gene was identified in 9 (81.8%) of 11 trimethoprim/sulfamethoxazole-resistant isolates, and the cmlA gene was identified in only 2 (18.1%) among 11 chloramphenicol-resistant isolates. Eighteen (75.0%) of the 24 MDR Salmonella were identified as containing class 1 integrons, within which dfrA12-aadA2 (55.6%) was the most prevalent resistance gene cassettes. Twenty-one (87.5%) of the MDR isolates were also found to have the plasmid replicons. Replicon B/O (41.7%) was the most prevalent replicon types. These results suggest that edible pork offal can become a reservoir that not only harbors MDR Salmonella, but also contributes to their dissemination through cross-contamination processes.

Clonal Structure and Antibiotic Resistance of Enterococcus spp. from Wild Birds in Poland.

The purpose of this study was to analyze the antibiotic resistance and genetic diversity of 27 enterococci (Enterococcus faecium, Enterococcus hirae, Enterococcus durans, and Enterococcus casseliflavus) isolated from wild bird species. Resistance to lincomycin was most common, followed by erythromycin, ciprofloxacin, tetracyclines, high level of aminoglycoside, and β-lactam antibiotics. No vancomycin- and chloramphenicol-resistant isolates were identified. The antibiotic resistance was linked to the tet(M), tet(L), erm(A), erm(B), msr(A/B), pbp5, ant(6)-Ia, and aph(3')-IIIa genes. Tn916/Tn1545-like transposons were detected. The high-level resistance to gentamicin was associated with the presence of gene aph(2″)-Id. All 18 E. faecium isolates were divided into 16 pulsotypes and 17 sequence types (STs), among which 7 STs were newly assigned (ST1266-ST1272). A majority of E. faecium isolates possess multilocus sequence typing profiles belonging to clonal complex 17 (CC17), the major epidemic lineage responsible for nosocomial infections. Two ST17 and newly described ST1267 and ST1271 (an SLV and DLV of ST17, respectively) of E. faecium isolates carried the type 1 allele of the housekeeping gene purK detected in hospital-related strains. Our results indicated that wild birds could be a source of resistant E. faecium isolates, belonging to CC17 and may represent a hazard to human health by transmission of these isolates.

Detection of Escherichia coli Producing CTX-M-1-Group Extended-Spectrum β-Lactamases from Pigs in Aichi Prefecture, Japan, between 2015 and 2016.

We investigated the prevalence and characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from Japanese pigs. A total of 345 pig fecal specimens were collected from 30 farms in the Aichi prefecture of Japan between June 2015 and April 2016, and 22 unique ESBL-producing E. coli were isolated from 16 samples spanning 8 farms. The ESBL types included CTX-M-15 (54.5%), CTX-M-55 (27.2%), CTX-M-3 (0.9%), and CTX-M-14 (0.9%). The predominant plasmid replicon type was IncN, and the isolates carried blaCTX-M-55. Nine sequence type (ST)s, including ST117, ST1706, ST38, and ST10, were detected in the ESBL-producers, but no B2-O25-ST131 was found. ESBL producers were highly resistant to cefotaxime, ceftiofur, and tetracycline, but were susceptible to imipenem, amikacin, and fosfomycin (FOM), although 2 ST354 isolates showed resistance to ciprofloxacin. All 11 chloramphenicol-resistant isolates, including ST117 (n = 6) and ST38 (n = 3) isolates, harbored floR, and the 2 FOM-resistant ST38 isolates harbored fosA3. Our results suggest that pigs do not act as direct reservoirs in the transmission of ESBL genes to E. coli in humans. However, ST117 E. coli carrying IncN-type plasmids mediating blaCTX-M-55 were isolated from several different farms, suggesting the potential for future spread in Japan. Therefore, plasmid sequence analyses and continuous surveillance are necessary from an epidemiological point of view and are required to better protect against ESBL-producer transmission.

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety

ABSTRACTFoodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.

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.

Molecular Epidemiology of Streptococcus pneumoniae Isolates from Children with Recurrent Upper Respiratory Tract Infections.

A total of 125 isolates were recovered from adenoids and/or nasopharynx of 170 children aged 2 to 5 from south-east Poland; they had undergone adenoidectomy for recurrent and/or persistent symptoms of upper respiratory tract infections. Pneumococcal isolates were analyzed by phenotyping (serotyping and antimicrobial resistance tests) and genotyping together with the clonality of the pneumococcal isolates based on resistance determinants, transposon distribution and multilocus sequence typing (MLST). Serotypes 19F, 6B and 23F constituted 44.8% of the isolates. Among all of the strains, 44.8% showed decreased susceptibility to penicillin and resistance to co-trimoxazole (52.8%), tetracycline (38.4%), erythromycin (53.6%), clindamycin (52.8%) and chloramphenicol (27.2%) was observed. Tn6002 was found in 34.8% of erythromycin-resistant isolates while composite Tn2010—in 16.7% of erm(B)-carrying isolates that harboured also mef(E) gene. Tn3872-related elements were detected in 27.3% of erythromycin-resistant strains. In the majority of chloramphenicol-resistant catpC194-carrying isolates (79.4%), ICESp23FST81-family elements were detected. The genotyping showed that pneumococcal population was very heterogeneous; 82 sequence types (STs) were identified, and the most frequent contributed to not more than 8% of the isolates. Nearly 44% STs were novel, each of them was recovered only from one child. Four STs belonged to one of the 43 worldwide spread resistant pneumococcal clones currently accepted by Pneumococcal Molecular Epidemiology Network (PMEN), i.e. Spain 9V-3, Spain 23F-1, Norway NT-42 and Poland 6B-20, accounting for 12 (16.7%) of the 75 nonususceptible isolates, and five STs were single-locus variants of PMEN resistant clones (England 14–9, Spain 9V-3, Spain 23F-1, Greece 21–30, Denmark 14–32), accounting 9 (12%) of nonsusceptible isolates. A few MDR clones belonging to 6B and 19F serotypes found among preschool children emphasizes rather the role of clonal dissemination of local strains in the community than international clones spreading in the increase of resistance among pneumococcal strains.

A meta-analysis of the proportion of animal Salmonella isolates resistant to drugs used against human salmonellosis in Ethiopia.

BackgroundThe emergence and spread of drug resistant Salmonellae of both human and animal origins are global concerns and worrisome in countries where the risk of infection is high and treatment options are limited. The objective of this study was to estimate the proportions of animal isolates resistant to antimicrobials used against human salmonellosis in Ethiopia.MethodsPublished studies on the antimicrobial resistance features of Salmonellae isolated from food animals of Ethiopia were searched in Medline, Google Scholar and the lists of references of articles. Eligible studies were selected by using inclusion and exclusion criteria and data were extracted. The extracted data included the host species, the numbers of isolates and the numbers of ampicillin, co-trimoxazole, chloramphenicol, ceftriaxone and ciprofloxacin resistant isolates. The risks of bias were assessed and the percentages of the variations of the estimates attributable to heterogeneities were quantified. Pooled proportions were estimated by the DerSimonian and Laird random effects model.ResultsFive hundred and fifty four Salmonellae isolated from cattle, camels, sheep, goats and pigs were tested with a variety of antimicrobials. The percentages of the variations attributable to heterogeneities were low for chloramphenicol and ceftriaxone (I2 = 0) and high for ampicillin, co-trimoxazole and ciprofloxacin resistance estimates (I2 > 75%). The pooled estimate of ampicillin resistant isolates was higher in slaughtered ruminants (17.28%) than in pigs (3.95%), (p < 0.001). The pooled estimates of co-trimoxazole resistant isolates in true ruminants (4.35%) and pigs (1.12%) were not significantly different (p > 0.05). The overall pooled estimates of chloramphenicol and ceftriaxone resistant isolates were 2.24% and 1.25%, respectively. Seven serotypes have been reported to be resistant to antimicrobials uncommonly used in veterinary clinical practice in Ethiopia.ConclusionsAmong Salmonellae of farm animals, there exist strains that are resistant to drugs used in the therapeutic management of human salmonellosis in Ethiopia. Intervention measures should be taken to ensure the prudent use of antimicrobials and curb the spread of high risk strains across the country.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0835-x) contains supplementary material, which is available to authorized users.

A meta-analysis of the proportion of antimicrobial resistant human Salmonella isolates in Ethiopia.

BackgroundAntimicrobial resistant Salmonella is a global problem and recently, a strain on the verge of pan-resistance was reported. In Ethiopia, the therapeutic management of Salmonellosis is difficult because drug sensitivity tests are not routinely carried out and treatment alternatives are not available in most health care facilities. The objectives of this study were to estimate the temporal changes and proportions of drug resistant isolates in Ethiopia.MethodsPublished studies on drug resistant Salmonella isolates were searched in Medline, Google Scholar and the lists of references of articles. Eligible studies were selected by using inclusion and exclusion criteria. Generic, methodological and statistical information were extracted from the eligible studies. The extracted data included the proportions of ampicillin, co-trimoxazole, chloramphenicol, ceftriaxone, ciprofloxacin and multi-drug resistant isolates. Pooled proportions were estimated by a random effects model.ResultsThe odds of multi-drug resistant isolates in the 2000s was higher than before the 1990s (OR =18.86, 95% CI = 13.08, 27.19). The pooled proportions of ampicillin, co-trimoxazole, chloramphenicol, ciprofloxacin and multi-drug resistant isolates in the 2000s were 86.01%, 68.01%, 62.08%, 3.61% and 79.56% respectively. S. Concord (>97%) was resistant to ampicillin, co-trimoxazole, chloramphenicol and ceftriaxone.ConclusionThe proportion of drug resistant isolates has increased since the 1970s. All drugs currently used for the treatment of Salmonellosis but ciprofloxacin are not reliable for an empirical therapy. Alternative drugs should be included in the essential drug list and measures should be taken to re-enforce the drug use policy.

The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.

Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci

To investigate the distribution of erythromycin, tetracycline and chloramphenicol resistance mechanisms and determinants and the relevant genetic environments and elements in viridans group streptococci (VGS). A total of 263 VGS collected from routine throat swabs in 2010-12 and identified to the species level were studied. Antibiotic resistance determinants and the relevant genetic contexts and elements were determined using amplification and sequencing assays and restriction analysis. The investigation provided original information on the distribution of resistance mechanisms, determinants and genetic elements in VGS. Erythromycin-resistant isolates totalled 148 (56.3%; 37 belonging to the cMLS phenotype and 111 belonging to the M phenotype); there were 72 (27.4%) and 7 (2.7%) tetracycline- and chloramphenicol-resistant isolates, respectively. A number of variants of known genetic contexts and elements carrying determinants of resistance to these antibiotics were detected, including the mega element, Φ10394.4, Tn2009, Tn2010, the IQ element, Tn917, Tn3872, Tn6002, Tn916, Tn5801, a tet(O) fragment from ICE2096-RD.2 and ICESp23FST81. These findings shed new light on the distribution of antibiotic resistance mechanisms and determinants and their genetic environments in VGS, for which very few such data are currently available. The high frequency and broad variety of such elements supports the notion that VGS may be important reservoirs of resistance genes for the more pathogenic streptococci. The high rates of macrolide resistance confirm the persistence of a marked prevalence of resistant VGS in Europe, where macrolide resistance is, conversely, declining among the major streptococcal pathogens.

Wild birds as biological indicators of environmental pollution: antimicrobial resistance patterns of Escherichia coli and enterococci isolated from common buzzards (Buteo buteo)

A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard faecal samples. The E. coli isolates showed high levels of resistance to streptomycin and tetracycline. The following resistance genes were detected: bla(TEM) (20 of 22 ampicillin-resistant isolates), tet(A) and/or tet(B) (16 of 27 tetracycline-resistant isolates), aadA1 (eight of 27 streptomycin-resistant isolates), cmlA (three of 15 chloramphenicol-resistant isolates), aac(3)-II with/without aac(3)-IV (all seven gentamicin-resistant isolates) and sul1 and/or sul2 and/or sul3 [all eight sulfamethoxazole/trimethoprim-resistant (SXT) isolates]. intI1 and intI2 genes were detected in four SXT-resistant isolates. The virulence-associated genes fimA (type 1 fimbriae), papC (P fimbriae) and aer (aerobactin) were detected in 61.1, 13.8 and 11.1% of the isolates, respectively. The isolates belonged to phylogroups A (47.2%), B1 (8.3%), B2 (13.9%) and D (30.5%). For the enterococci isolates, Enterococcus faecium was the most prevalent species (48.4%). High levels of tetracycline and erythromycin resistance were found among our isolates (87 and 81%, respectively). Most of the tetracycline-resistant strains carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 80% of erythromycin-resistant isolates. The vat(D) and/or vat(E) genes were found in nine of the 17 quinupristin-dalfopristin-resistant isolates. The enterococcal isolates showing high-level resistance for kanamycin, gentamicin and streptomycin contained the aph(3')-IIIa, aac(6')-aph(2″) and ant(6)-Ia genes, respectively. This report reveals that common buzzards seem to represent an important reservoir, or at least a source, of multi-resistant E. coli and enterococci isolates, and consequently may represent a considerable hazard to human and animal health by transmission of these isolates to waterways and other environmental sources via their faecal deposits.

Comparison of the Prevalences and Antimicrobial Resistances of Escherichia coli Isolates from Different Retail Meats in the United States, 2002 to 2008

Escherichia coli isolates were recovered from the National Antimicrobial Resistance Monitoring System retail meat program and examined for antimicrobial susceptibility. Retail meat samples (n = 11,921) from four U.S. states collected during 2002 to 2008, consisting of 2,988 chicken breast, 2,942 ground turkey, 2,991 ground beef, and 3,000 pork chop samples, were analyzed. A total of 8,286 E. coli isolates were recovered. The greatest numbers of samples contaminated with the organism were chicken (83.5%) and turkey (82.0%), followed by beef (68.9%) and pork (44.0%). Resistance was most common to tetracycline (50.3%), followed by streptomycin (34.6%), sulfamethoxazole-sulfisoxazole (31.6%), ampicillin (22.5%), gentamicin (18.6%), kanamycin (8.4%), amoxicillin-clavulanic acid (6.4%), and cefoxitin (5.2%). Less than 5% of the isolates had resistance to trimethoprim, ceftriaxone, ceftiofur, nalidixic acid, chloramphenicol, and ciprofloxacin. All isolates were susceptible to amikacin. Compared to beef and pork isolates, the poultry meat isolates had a greater percentage of resistance to all tested drugs, with the exception of chloramphenicol, to which pork isolates had the most resistance. More than half of the turkey isolates (56%) were resistant to multidrugs (≥3 classes) compared to 38.9% of chicken, 17.3% of pork, and 9.3% of beef isolates. The bla(CMY) gene was present in all ceftriaxone- and ceftiofur-resistant isolates. The cmlA, flo, and catI genes were present in 45%, 43%, and 40% of chloramphenicol-resistant isolates, respectively. Most nalidixic acid-resistant isolates (98.5%) had a gyrA mutation in S83 or D87 or both, whereas only 6.7% had a parC mutation in either S80 or E84. The results showed that E. coli was commonly present in the retail meats, and antimicrobial resistance profiles differed according to the animal origin of the isolates.

Serotypes, Virulence Genes, and Antimicrobial Susceptibility ofEscherichia coliIsolates from Pigs

One hundred two pathogenic Escherichia coli isolates from diseased pigs were analyzed for serotypes, virulence genes, antimicrobial susceptibility, and the molecular basis of phenicol resistance. Of these 102 E. coli isolates, 101 were typeable and belonged to 27 different O serogroups. However, 69% of these isolates belonged to one of the following eight serogroups: O8, O54, O64, O65, O92, O108, O119, and O120. Serogroups O8 (23%) and O64 (10%) were the most prevalent among typeable isolates. High-resistance phenotypes were observed in all the isolates, with the majority displaying resistance to chloramphenicol (89%), streptomycin (83%), enrofloxacin (78%), and doxycycline (60%). The chloramphenicol resistance genes cat1, cat2, and cmlA were detected in 58%, 49%, and 65%, respectively, of the chloramphenicol-resistant isolates. The floR gene was detected in 57% of the florfenicol-resistant isolates and in 52% of chloramphenicol-resistant isolates. Pulsed-field gel electrophoresis showed that the 32 floR-positive isolates with florfenicol minimum inhibitory concentration ≥ 8 μg mL⁻¹ belonged to 25 different pulsed-field gel electrophoresis profiles, indicating the spread of floR among swine pathogenic E. coli isolates.