Genotypic Antimicrobial Resistance Research Articles

Genetic lineages and antimicrobial resistance genotypes in Staphylococcus aureus from children with atopic dermatitis: detection of clonal complexes CC1, CC97 and CC398

The objective was to analyse the genetic lineages of Staphylococcus aureus recovered from nasal and skin samples of atopic dermatitis (AD) paediatric patients, and to characterize the antimicrobial resistance phenotype–genotype and the immune-evasion-cluster (IEC) type of isolates. Forty S. aureus isolates from 35 patients (skin: 26; nasal samples: 14) were characterized. Isolates were submitted to spa-, agr- and multilocus sequence typing. All S. aureus strains analyzed were methicillin-susceptible (MSSA). High genetic diversity was detected among the 40 MSSA isolates (especially among skin isolates), with detection of 27 different spa-types, 20 sequence-types and 16 clonal complexes (CCs). Lineages CC30 and CC5 were predominant among nasal isolates (71% vs 23% skin). Thirteen different CCs were detected among skin isolates, with detection of clades CC1, CC9 and CC398. Antimicrobial resistance rates detected were higher in skin than in nasal isolates, especially for macrolides, aminoglycosides, lincosamides and mupirocin. MSSA strains were characterized into five IEC-types, being A, B and F the predominant ones. MSSA strains of lineages CC45 and CC5 were detected in almost all cases in AD patients with severe Scoring Atopic Dermatitis (SCORAD) and lineages CC8, and CC30 in those with mild or moderate one. As conclusion, high-clonal-diversity was detected among MSSA from AD patients, especially in skin-isolates. Colonization with S. aureus of some CCs seems more associated with AD severity than other lineages.

Longitudinal characterization of monophasic Salmonella Typhimurium throughout the pig’s life cycle

Swine have been described as an important reservoir of multidrug resistant monophasic Salmonella Typhimurium, though information on its ecology is scarce. A longitudinal study was performed in order to elucidate the Salmonella 4,[5],12:i:- dynamics throughout the pig’s production cycle. A total of 209 faecal samples were collected from 10 sows and in six sampling times during the life of 70 pigs from a Portuguese industrial farm, and 43 isolates of S. 4,[5],12:i:- were identified and characterized regarding clonality and antimicrobial resistance phenotype and genotype. Most isolates (n=42) exhibited resistance to at least ampicillin, kanamycin, neomycin, streptomycin, tetracycline and sulfonamides (encoded by blaTEM, aphAI-IAB, strA, strB, tetB and sul2, respectively). Isolates obtained during the finishing phase showed additional resistance to chloramphenicol and florfenicol (floR), gentamicin and netilmicin (aac(3′)-IV). To our knowledge, this study is the first description of aphAI-IAB in S. 4,[5],12:i:-. PFGE analysis showed uneven distribution of isolates into three clusters, A (n=34), B (n=8) and C (n=1). PFGE cluster A was predominant in sows (n=5) and piglets in the farrowing phase (n=17) and in pigs in the early finishing phase (n=11) suggesting a carryover from birth to adult age. The introduction of PFGE cluster B isolates in adulthood could have had an external source, reinforcing the relevance of environmental transmission in the farm ecosystem. This study reveals a dynamic interaction between monophasic S. Typhimurium and the pressures exerted under an intensive swine production setting.

Antimicrobial Resistance and Resistance Genes in Aerobic Bacteria Isolated from Pork at Slaughter

The aim of this study was to investigate the phenotypic and genotypic antimicrobial resistance, integrons, and transferability of resistance markers in 243 aerobic bacteria recovered from pork at slaughter in the People's Republic of China. The organisms belonged to 22 genera of gram-negative bacteria (92.2%) and gram-positive bacteria (7.8%). High levels of resistance were detected to tetracycline, trimethoprim-sulfamethoxazole, and ampicillin (36.2 to 54.3%), and lower levels were detected to nitrofurantoin, cefotaxime, gentamicin, ciprofloxacin, and chloramphenicol (7.8 to 29.2%). Across species, genes conferring antimicrobial resistance were observed with the following frequencies: blaTEM, 40.7%; blaCMY-2, 15.2%; blaCTX-M, 11.5%; sul2, 27.2%; sul1, 14.4%; tet(A), 5.4%; tet(L), 5.4%; tet(M), 5.0%; tet(E), 3.7%; tet(C), 3.3%; tet(S), 2.5%; and tet(K), 0.8%. Various antimicrobial resistance genes were found in new carriers: blaTEM in Lactococcus garvieae, Myroides odoratimimus, Aeromonas hydrophila, Staphylococcus sciuri, Raoultella terrigena, Macrococcus caseolyticus, Acinetobacter ursingii, Sphingobacterium sp., and Oceanobacillus sp.; blaCMY-2 in Lactococcus lactis, Klebsiella oxytoca, Serratia marcescens, Acinetobacter baumannii, and Myroides phaeus; tet(L) in M. caseolyticus; sul1 in Vibrio cincinnatiensis; sul2 in Acinetobacter bereziniae, Acinetobacter johnsonii, and V. cincinnatiensis; and the class 1 integron and gene cassette aadA2 in V. cincinnatiensis. Approximately 6.6% of isolates contained class 1 integrons, and one isolate harbored class 2 integrons. Plasmid associated intI1 and androgen receptor- encoding genes were transferred into Escherichia coli J53 and E. coli DH5α by conjugation and transformation experiments, respectively. Our study highlights the importance of aerobic bacteria from pork as reservoirs for antimicrobial resistance genes and mobile genetic elements that can readily be transferred intra- and interspecies.

High prevalence of Staphylococcus haemolyticus and Staphylococcus saprophyticus in environmental samples of a Tunisian hospital

The purpose of this study was to evaluate the rate of detection of coagulase negative staphylococci (CoNS) in environmental samples of 17 services in a Tunisian hospital, determining the antimicrobial resistance phenotypes and genotypes of recovered isolates. To our knowledge, this is the first study that determines the prevalence of CoNS with correlation of antibiotic resistance in the hospital environment in Tunisia. CoNS were obtained from 83 of the 200 tested samples (41.5%). Staphylococcus haemolyticus was the most prevalent species (45.8%), followed by S. saprophyticus (36.1%). The remaining CoNS species detected were S. epidermidis, S. cohnii, S. warneri, S. sciuri, S. simulans, S. pasteuri, S. arlettae, and S. xilosus. Methicillin-resistant CoNS were detected in 20 of the 200 tested samples (10%), and the mecA gene was demonstrated in 18 S. haemolyticus, one S. epidermidis and one S. saprophyticus isolates. Methicillin susceptible isolates were detected in 63 samples (31.5%). Antimicrobial resistance genes detected were as follows (number of isolates): erythromycin [msr(A) (n = 32); erm(C) (n = 8)], tetracycline [tet(K) and/or tet(M) (n = 21)], gentamicin [aac(6′)-Ie-aph(2″)-Ia (n = 16)], kanamycin [(aph(3′)-IIIa (n = 19)], tobramycin [ant(4′)-Ia (n = 14)], and streptomycin [ant(6′)-Ia (n = 3)]. The high frequency of detection of multi-drug-resistant CoNS in the hospital environment, especially S. haemolyticus and S. saprophyticus, is of relevance and could be due to cross-transmission between patients, staff, and environment.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolated from Australian Veterinarians.

This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3–92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2–532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.

Conjugative IncF and IncI1 plasmids with tet(A) and class 1 integron conferring multidrug resistance in F18(+) porcine enterotoxigenic E. coli.

Enterotoxigenic E. coli (ETEC) bacteria frequently cause watery diarrhoea in newborn and weaned pigs. Plasmids carrying genes of different enterotoxins and fimbrial adhesins, as well as plasmids conferring antimicrobial resistance are of prime importance in the epidemiology and pathogenesis of ETEC. Recent studies have revealed the significance of the porcine ETEC plasmid pTC, carrying tetracycline resistance gene tet(B) with enterotoxin genes. In contrast, the role of tet(A) plasmids in transferring resistance of porcine ETEC is less understood. The objective of the present study was to provide a comparative analysis of antimicrobial resistance and virulence gene profiles of porcine post-weaning ETEC strains representing pork-producing areas in Central Europe and in the USA, with special attention to plasmids carrying the tet(A) gene. Antimicrobial resistance phenotypes and genotypes of 87 porcine ETEC strains isolated from cases of post-weaning diarrhoea in Austria, the Czech Republic, Hungary and the Midwest USA was determined by disk diffusion and by PCR. Central European strains carrying tet(A) or tet(B) were further subjected to molecular characterisation of their tet plasmids. Results indicated that > 90% of the ETEC strains shared a common multidrug resistant (MDR) pattern of sulphamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. Tetracycline resistance was most frequently determined by the tet(B) gene (38%), while tet(A) was identified in 26% of all isolates with wide ranges for both tet gene types between some countries and with class 1 integrons and resistance genes co-transferred by conjugation. The virulence gene profiles included enterotoxin genes (lt, sta and/or stb), as well as adhesin genes (k88/f4, f18). Characterisation of two representative tet(A) plasmids of porcine F18(+) ETEC from Central Europe revealed that the IncF plasmid (pES11732) of the Czech strain (~120 kb) carried tet(A) in association with catA1 for chloramphenicol resistance. The IncI1 plasmid (pES2172) of the Hungarian strain (~138 kb) carried tet(A) gene and a class 1 integron with an unusual variable region of 2,735 bp composed by two gene cassettes: estX-aadA1 encoding for streptothricin-spectinomycin/streptomycin resistance exemplifying simultaneous recruitment, assembly and transfer of multidrug resistance genes by the tet(A) plasmid of porcine ETEC. By this we provide the first description of IncF and IncI1 type plasmids of F18(+) porcine enterotoxigenic E. coli responsible for cotransfer of the tet(A) gene with multidrug resistance. Additionally, the unusual determinant estX, encoding for streptothricin resistance, is first reported here in porcine enterotoxigenic E. coli.

Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

The objectives of this study were to identify antimicrobial resistance genotypes for Campylobacter and to evaluate the correlation between resistance phenotypes and genotypes using in vitro antimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114 Campylobacter species isolates (82 C. coli and 32 C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, including tet(O), blaOXA-61, catA, lnu(C), aph(2″)-Ib, aph(2″)-Ic, aph(2')-If, aph(2″)-Ig, aph(2″)-Ih, aac(6')-Ie-aph(2″)-Ia, aac(6')-Ie-aph(2″)-If, aac(6')-Im, aadE, sat4, ant(6'), aad9, aph(3')-Ic, and aph(3')-IIIa, and mutations in two housekeeping genes (gyrA and 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs.

Prevalence, antimicrobial resistance and genetic diversity of Yersinia enterocolitica isolated from retail frozen foods in China.

In this study, our aim was to estimate the extent of Yersinia enterocolitica contamination in frozen foods in China and determine the bioserotype, virulotype, antimicrobial resistance, and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) genotyping profiles of recovered Y. enterocolitica isolates. Out of 455 samples collected between July 2011 and May 2014, 56 (12.3%) tested positive for Y. enterocolitica. The 70 isolated strains were grouped into five clusters and one singleton based on their ERIC-PCR fingerprint, at a similarity coefficient of 70%. All strains were of biotype 1A, and 35.7% were of bioserotype 1A/O:8. Most strains lacked the virulence genes ail, virF, ystA, and ystC, but harbored ystB, fepD, ymoA, fes and sat. All strains were sensitive to ticarcillin but resistant to two or more antibiotics, and 48.6% of the strains were resistant to four to nine antibiotics. High resistance rates were observed for ampicillin, cephalothin, trimethoprim/sulfamethoxazole, amoxicillin/clavulanic acid, nalidixic acid and chloramphenicol (98.6%, 95.7%, 74.3%, 28.6%, 18.6% and 12.9%, respectively). This study provides a systematic surveillance of Y. enterocolitica prevalence in frozen foods in China and indicates its high antibiotic resistance, which could serve as useful information for the government to control Y. enterocolitica contamination in frozen foods and the use of antibiotics.

Characterization of Antimicrobial Resistance in Salmonella enterica Isolated from Pork, Chicken Meat, and Humans in Northeastern Thailand.

A total of 221 Salmonella enterica from raw pork (n=64), raw chicken (n=80), and humans (n=77) were characterized for antimicrobial resistance phenotypes and genotypes and virulence plasmid-associated genes. Most Salmonella isolates (95.9%) were multidrug resistant and exhibited high resistance to sulfamethoxazole (96.4%), streptomycin (93.2%), spectinomycin (76.5%), tetracycline (73.3%), ampicillin (70.1%), and trimethoprim (60.2%). Forty-one percent of all isolates were intI1-positive, of which 60% carried class 1 integrons with variable region ranging in size from 0.2 to 2.0 kb. Six integron profiles (IP-I to IP-VI) were defined. The dfrA12-aadA2 cassette was most prevalent (66.7%). Class 1 integrons with the dfrA12-aadA2 cassette in five pork isolates could be horizontally transferred. Three pork isolates carried Salmonella genomic island 1 (SGI1), of which a serovar Anatum harbored SGI1 gene cluster located between thdF and int2. Two single-point mutations (i.e., G-259-T and C-248-T) in gyrA leading to Asp-87-Tyr and Ser-83-Phe substitutions in GyrA, respectively, were detected. Of all plasmid-mediated quinolone resistance genes tested, only qnrS (4.1%) and qnrB (1.8%) were found. The virulence plasmid-associated genes including spvC, pefA, and rck were identified in 8.1%, 1.8%, and 1.4% of all Salmonella isolates, respectively.

Characterization of fecal vancomycin-resistant enterococci with acquired and intrinsic resistance mechanisms in wild animals, Spain.

The objectives were to evaluate the presence of vancomycin-resistant enterococci with acquired (VRE-a) and intrinsic (VRE-i) resistance mechanisms in fecal samples from different wild animals, and analyze their phenotypes and genotypes of antimicrobial resistance. A total of 348 cloacal/rectal samples from red-legged partridges (127), white storks (81), red kites (59), and wild boars (81) (June 2014/February 2015) were inoculated in Slanetz-Bartley agar supplemented with vancomycin (4μg/mL). We investigated the susceptibility to 12 antimicrobials and the presence of 19 antimicrobial resistance and five virulence genes. In addition, we performed multilocus sequence typing, detection of IS16 and studied Tn1546 structure. One VRE-a isolate was identified in one wild boar. This isolate was identified as Enterococcus faecium, harbored vanA gene included into Tn1546 (truncated with IS1542/IS1216), and belonged to the new ST993. This isolate contained the erm(A), erm(B), tet(M), dfrG, and dfrK genes. Neither element IS16 nor the studied virulence genes were detected. Ninety-six VRE-i isolates were identified (89 Enterococcus gallinarum and seven Enterococcus casseliflavus), with the following prevalence: red kites (71.2%), white storks (46.9%), red-legged partridges (7.9%), and wild boars (4.9%). Most E. gallinarum isolates showed resistance to tetracycline (66.3%) and/or erythromycin (46.1%). High-level resistance to aminoglycosides was present among our VRE-i isolates: kanamycin (22.9%), streptomycin (11.5%), and gentamicin (9.4%). In general, VRE-i isolates of red kites showed higher rates of resistance for non-glycopeptide agents than those of other animal species. The dissemination of acquired resistance mechanisms in natural environments could have implications in the global spread of resistance with public health implications.

Antimicrobial resistance genotypes and phenotypes from multidrug-resistant bacterial wound infection isolates in Cambodia.

This study aimed to identify the molecular determinants responsible for antibiotic resistance among human wound isolates in Cambodia. Staphylococcus spp. (n=10) and a variety of Gram-negative isolates (n=21) were taken from a larger collection of wound isolates collected during 2011-2013 and were analysed for the presence of >230 resistance determinants using a broad-spectrum DNA microarray. These isolates were chosen to represent the species most commonly found in wound isolates referred during this time and to include some of the most resistant strains. Resistance determinants detected among the staphylococci included blaZ (90%), mecA (100%), erm(B) (70%), erm(C) (20%), tet(38) (90%), tet(K) (40%), tet(Lp) (10%), tet(M) (20%), lnu(A)/lin(A) and lnu(B)/lin(B) (10% each), msr(A)/msr(B)/msr(SA) (10%), norA (80%) and dfrA (10%). Eleven different β-lactamase genes were detected among the Gram-negative bacteria, including genes encoding the TEM (48%), CTX-M-1 (48%), CTX-M-9 (5%), SHV (5%) and VEB (10%) families of broad-spectrum and extended-spectrum β-lactamase enzymes, as well as the carbapenemase gene blaOXA-23. Forty additional genes were also detected in the Gram-negative isolates conferring resistance to aminoglycosides (11 genes), phenicols (5 genes), macrolides [4 genes, including mph(A)/mph(K) (10%)], lincosamides [lnu(F)/lin(F), lnu(G)/lin(G)], tetracycline (4 genes), rifampicin [arr (29%)], quaternary amines [qacEΔ1 (43%)], quinolones [qnrS (14%) and qnrB (5%)], sulfonamides [sul1 (29%), sul2 (38%) and sul3 (10%)], streptothricin (sat2) and trimethoprim (6 genes). The results obtained here provide a snapshot of the broad variety of resistance determinants currently circulating within Cambodia.

Studies on the drug-resistance genes and its epidemiology of the strains produced extended spectrum β-lactamases of Klebsiella neumonia in Pediatric Intensive Care Unit

Objective To explore the antimicrobial resistance genotypes and molecular epidemic features of Klebsiella neumonia (K.pneumoniae) producing extended spectrum β-lactamases (ESBLs) in the Pediatric Intensive Care Unit (PICU) of Guiyang Children's Hospital. Methods Disc diffusion technique (Kirby-Bauer method) and automatic microbiology analysis system were employed to determine the antimicrobial resistance, and Double-disk Diffusion was adopted in the phenotype confirmatory test of ESBLs, and PCR was used to determine the antimicrobial resistance genotypes. Results Among 44 straits of non-repetitive-K.pneumoniae, isolated from the children during hospitalization since April to December of 2013, 29 straits(65.9%) were detected.The findings of sensitivity tests showed that 29 strains of ESBLs-producing K. pneumoniae presented a higher rate of sensitivity to carbapenems, cephamycin and quinolones, 100% resistance to penicillin and cephalosporins of the first and the second generations.Fifteen non-ESBLs-producing K. pneumoniae presented 100% resistance to penicillin.The rate of resistance to 9 kinds of antibiotics(Ampicillin/Sulbactam, Cefazolin, Cefuroxime, Cefamandole, Ceftriaxone, Ceftazidime, Cefepime, Gentamicin, Aztreonam) in ESBLs-producing K. pneumoniae strains(79.3%, 100.0%, 100.0%, 100.0%, 100.0%, 79.3%, 65.5%, 41.4%, 79.3%) was significantly higher than that of non-ESBLs-producing K. pneumoniae trains(13.3%, 6.7%, 20.0%, 20.0%, 0, 0, 0, 6.7%, 0) (χ2=17.54, 35.51, 28.00, 28.00, 44.00, 24.93, 17.30, 4.18, 24.93, all P<0.05=. In 29 strains of ESBLs-producing K. pneumoniae, 3 genotypes were detected respectively: 93.1% of SHV (27/29 strains), 51.7% of TEM (15/29 strains) and 37.9% of CTX-M (11/29 strains). Five forms of genotype distribution were presented: 14 (43.8%) strains carrying single ESBLs gene, 5 (17.2%) strains carrying 2 types, 19 (31.0%) strains carrying 3 types, and 1 strain had not been genotyped. Conclusions ESBLs-producing K. pneumoniae had been epidemic in PICU of Guiyang Children's Hospital, with multiple antimicrobial resistances and diversification of antimicrobial resistance genotypes. Key words: Intensive Care Unit; Extended spectrum β-lactamase; Klebsiella pneumoniae; Genotype; Child

Mismatch between antimicrobial resistance phenotype and genotype of pathogenic Vibrio parahaemolyticus isolated from seafood

Antimicrobial resistance phenotypes (18 antimicrobials; disk diffusion method) and genotypes (38 antimicrobial resistance genes; PCR) of 20 pathogenic Vibrio parahaemolyticus strains isolated from seafood in Shanghai wholesale markets between 2009 and 2013 were evaluated. Seventeen isolates (85%) were resistant to one or more antimicrobials, and highest resistance was observed to ampicillin (85%) and cephazolin (30%). And the isolates with tdh displayed higher resistant rates than isolates with trh. Eight antimicrobial resistance genes (strB, aadA2, strA, tetA, floR, sulI, sulII, and sulIII) were detected in these isolates. Surprisingly, the antimicrobial resistance phenotypes and genotypes of these isolates were not consistent: some isolates were resistant to β-lactam or aminoglycoside, whereas the corresponding genes were negative. Comparatively, aminoglycoside, tetracycline and chloramphenicol resistance genes occurred in susceptibility isolates. This research reveals the mismatch phenomenon between the antimicrobial resistance phenotype and genotype of seafood-derived pathogenic V. parahaemolyticus, and that susceptibility isolates might be a potential risk source for storage and transmission of resistance genes.

Antimicrobial and disinfectant resistance of Escherichia coli isolated from giant pandas.

The study aims to demonstrate the antimicrobial and disinfectant resistance phenotypes and genotypes of Escherichia coli isolates obtained from giant pandas (Ailuropoda melanoleuca). Antimicrobial testing was performed according to the standard disk diffusion method. The minimal inhibitory concentrations (MICs) of disinfectants were determined using the agar dilution method. All isolates were screened for the presence of antimicrobial and disinfectant resistance genes and further analysed for genetic relatedness by pulse-field gel electrophoresis (PFGE). Results showed that 46·6% of the isolates were resistant to at least one antimicrobial. Escherichia coli isolates showed resistance to fewer antimicrobials as panda age increased. Among antimicrobial-resistant E. coli isolates, the antimicrobial resistance genes blaCTX-M (88·2%) and sul1 (92·3%) were most prevalent. The disinfectant resistance genes emrE, ydgE/ydgF, mdfA and sugE(c) were commonly present (68·2-98·9%), whereas qac and sugE(p) were relatively less prevalent (0-21·3%). The frequencies of resistance genes tended to be higher in E. coli isolated in December than in July, and PFGE profiles were also more diverse in isolates in December. The qacEΔ1 and sugE(p) genes were higher in adolescent pandas than in any other age groups. PFGE revealed that antimicrobial resistance correlated well with sampling time and habitat. This study demonstrated that antimicrobial and disinfectant resistance was common in giant panda-derived E. coli, and the antimicrobial resistance was associated with sampling time and habitat. Escherichia coli could serve as a critical vector in spreading disinfectant and antimicrobial resistance. This is the first study that demonstrated the phenotypic and genetic characterizations of antimicrobial and disinfectant resistance in E. coli isolates from more than 60 giant pandas. Frequent transfer of pandas to other cages may lead to the dissemination of antimicrobial resistance. The study highlights the need for regularly monitoring the antimicrobial and disinfectant resistance in bacteria from giant pandas.

Clonal lineages detected amongst tetracycline-resistant meticillin-resistant Staphylococcus aureus isolates of a Tunisian hospital, with detection of lineage ST398.

Tetracycline resistance has been postulated as a potential phenotypic marker of livestock-associated lineage ST398 amongst meticillin-resistant Staphylococcus aureus (MRSA) clinical isolates in some European hospitals. The objective of this study was to determine if this marker could also be applied to Maghrebian countries. In total, 99 MRSA isolates were collected in a Tunisian hospital during January 2011-October 2012, and 24 tetracycline-resistant MRSA isolates of this collection were characterized. All isolates were tested for antimicrobial resistance phenotypes and genotypes, molecular typing, and virulence genes. Multilocus sequence typing showed that the majority of the isolates (19/24) belonged to clonal complex CC8 (ST247, n = 12 isolates; ST239, n = 6 isolates; ST241, n = 1 isolate). The remaining isolates belonged to CC398 (ST398, n = 1 isolate), CC5 (ST5 and ST641, n = 2 isolates), and CC80 (ST728, n = 2 isolates). Spa typing discriminated MRSA in eight spa types: bib26 (n = 12 isolates), bib26 (n = 5 isolates), bib26 (n = 2 isolates), and bib26, bib26, bib26, bib26 and the new bib26 (n = 1 isolate each). Three agr groups were found amongst the studied isolates: agr group I (n = 20 isolates), agr group II (n = 2) and agr group III (n = 2 isolates). We report the detection of one MRSA ST398-t899 isolate in the nasal sample of a farmer patient in Tunisia, representing the first report of ST398 in humans in Africa. Tetracycline resistance seems not to be a good phenotypic marker for MRSA ST398 strains in Tunisia, where CC8 was the most prevalent lineage. Continuous efforts to understand the changing epidemiology of this micro-organism are necessary not only for appropriate antimicrobial treatment and effective infection control, but also to monitor its evolution.

Antimicrobial resistance determinants of Escherichia coli isolates recovered from some rivers in Osun State, South-Western Nigeria: Implications for public health

The inevitable development of resistance has sunk the great success achieved in the discovery of antimicrobial agents and dashed the hope of man in the recovery from infections and illnesses, as diseases and disease agents that were once thought to be controlled by antimicrobials are now re-emerging in new leagues resistance to therapy. A total of 300 PCR confirmed Escherichia coli isolates recovered from different river sources in Osun State, Nigeria were evaluated for their antibiogram profiling by the disc diffusion method and the resistant isolates were further profiled for their genotypic antimicrobial resistance determinants by polymerase chain reaction assays. Among the 20 antimicrobials selected from 10 families, resistance among sulfonamides, β-lactams and tetracyclines were found to be most frequent than phenicols and aminoglycosides with a noticeable increase in the number of multi-drug resistance ranging from three to nine antimicrobials. A total of 19 resistance determinants were assessed with their prevalence and distributions obtained as follows; [sulfonamides sulI (8%), sulII (41%)], [β-lactams; ampC 22%; blaTEM, (21%), and blaZ (18%),], [tetracyclines tetA (24%), tetB (23%), tetC (18%), tetD (78%), tetK (15%), and tetM, (10%)], [phenicols; catI (37%), catII (28%), and cmIA1 (19%)] and [aminoglycosides; aacC2 (8%), aphA1 (80%), aphA2 (80%), aadA (79%) and strA (38%)]. The Pearson chi-square exact test revealed many strong significant associations among ampC, blaTEM, blaZ and tetA genes with some determinants screened. The findings signify high increase in the prevalence of multidrug resistant E. coli isolates and resistance determinants indicating increased public health risks associated with the ingestion of waters from untreated sources. Hence, a necessity for safe water supply, provision of proper sanitation facilities and good surveillance programmes to monitor antimicrobial resistance patterns in water bodies.

Phenotypic and Genotypic Antimicrobial Resistance of Lactococcus Sp. Strains Isolated from Rainbow Trout (Oncorhynchus Mykiss)

Abstract A current profile of antimicrobial resistance and plasmid of 29 Lactococcus garvieae and one Lactococcus lactis strains isolated from rainbow trouts (Oncorhynchus mykiss) from farms throughout Turkey were investigated. All isolates were sensitive to penicillin G (90%), ampicillin (86.7%), florfenicol (83.3%), amoxicillin (80.1%), and tetracycline (73.4%), and resistant to trimethoprim+sulfamethoxazole (86.6%) and gentamycin (46.6%) by disc diffusion method. Twenty-eight (93%) isolates had two to seven antibiotic resistance genes (ARGs) determined by PCR. The most prevalent ARGs were tetracycline (tetB), erythromycin (ereB), and β-lactam (blaTEM). Bacterial strains were also screened for plasmid DNA by agarose gel electrophoresis and two strains harboured plasmids, with sizes ranging from 3 to 9 kb.

Molecular characterization of Staphylococcus aureus from nasal samples of healthy farm animals and pets in Tunisia.

A total of 261 healthy farm and pet animals (75 cattle, 52 goats, 100 dogs, and 34 cats) from different regions of Tunisia were screened for Staphylococcus aureus nasal carriage. Molecular typing of isolates (by spa- and multilocus sequence-typing) was performed, and their antimicrobial resistance and virulence genotypes were determined by PCR and sequencing. S. aureus isolates were detected in 17 of 261 tested samples (6.5%). All S. aureus isolates recovered were methicillin-susceptible (MSSA), and one isolate/sample was further studied. Eight different spa types were detected (t189, t279, t582, t701, t1166, t1268, t1534, and t1773), and eight different sequence types were identified (ST6, ST15, ST45, ST133, ST188, ST700 [clonal complex CC130], ST2057, and a new ST2121). MSSA from pets (six isolates) showed resistance to (number of isolates, resistance gene): penicillin (six, blaZ), tetracycline (one, tet[M]), erythromycin one, erm[A]), streptomycin (one, ant[6]-Ia), and ciprofloxacin (one). All isolates from farm animals showed susceptibility to the tested antimicrobials, except for two penicillin-resistant isolates. Five S. aureus isolates from goats and cats harbored the lukF/lukS-PV genes, encoding the Panton-Valentine leukocidin, and six isolates from goats harbored the tst virulence gene. In addition, diverse combinations of enterotoxin genes were detected, including two variants of the egc cluster. Goats and cats could represent a reservoir of important toxin genes, with potential implications in animal and human health.

Direct detection of markers associated with Neisseria gonorrhoeae antimicrobial resistance in New Zealand using residual DNA from the Cobas 4800 CT/NG NAAT assay

ObjectivesTo use nucleic acid amplification techniques (NAAT) for detection of markers associated with gonococcal antimicrobial resistance (AMR) in non-cultured clinical samples to enhance surveillance of Neisseria gonorrhoeae AMR in New...

Coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food of animal origin – Phenotypic and genotypic antibiotic resistance

The aim of this work was to study the pheno- and genotypical antimicrobial resistance profile of coagulase negative staphylococci (CoNS) isolated from 146 ready-to-eat food of animal origin (cheeses, cured meats, sausages, smoked fishes). 58 strains were isolated, they were classified as Staphylococcus xylosus (n = 29), Staphylococcus epidermidis (n = 16); Staphylococcus lentus (n = 7); Staphylococcus saprophyticus (n = 4); Staphylococcus hyicus (n = 1) and Staphylococcus simulans (n = 1) by phenotypic and genotypic methods. Isolates were tested for resistance to erythromycin, clindamycin, gentamicin, cefoxitin, norfloxacin, ciprofloxacin, tetracycline, tigecycline, rifampicin, nitrofurantoin, linezolid, trimetoprim, sulphamethoxazole/trimethoprim, chloramphenicol, quinupristin/dalfopristin by the disk diffusion method. PCR was used for the detection of antibiotic resistance genes encoding: methicillin resistance – mecA; macrolide resistance – erm(A), erm(B), erm(C), mrs(A/B); efflux proteins tet(K) and tet(L) and ribosomal protection proteins tet(M). For all the tet(M)-positive isolates the presence of conjugative transposons of the Tn916–Tn1545 family was determined. Most of the isolates were resistant to cefoxitin (41.3%) followed by clindamycin (36.2%), tigecycline (24.1%), rifampicin (17.2%) and erythromycin (13.8%). 32.2% staphylococcal isolates were multidrug resistant (MDR). All methicillin resistant staphylococci harboured mecA gene. Isolates, phenotypic resistant to tetracycline, harboured at least one tetracycline resistance determinant on which tet(M) was most frequent. All of the isolates positive for tet(M) genes were positive for the Tn916–Tn1545 -like integrase family gene. In the erythromycin-resistant isolates, the macrolide resistance genes erm(C) or msr(A/B) were present. Although coagulase-negative staphylococci are not classical food poisoning bacteria, its presence in food could be of public health significance due to the possible spread of antibiotic resistance.