Antibiotic Resistance Gene Cassettes Research Articles

Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain

The aim of this study was to determine the presence of virulence genes and antibiotic resistance profiles in 164 Escherichia coli strains isolated from birds (feral pigeons, hybrid ducks, house sparrows and spotless starlings) inhabiting urban and rural environments. A total of eight atypical enteropathogenic E. coli strains were identified: one in a house sparrow, four in feral pigeons and three in spotless starlings. Antibiotic resistance was present in 32.9% (54) of E. coli strains. The dominant type of resistance was to tetracycline (21.3%), ampicillin (19.5%) and sulfamethoxazole (18.9%). Five isolates had class 1 integrons containing gene cassettes encoding for dihydrofolate reductase A (dfrA) and aminoglycoside adenyltransferase A (aadA), one in a feral pigeon and four in spotless starlings. To our knowledge, the present study constitutes the first detection of virulence genes from E. coli in spotless starlings and house sparrows, and is also the first identification worldwide of integrons containing antibiotic resistance gene cassettes in E. coli strains from spotless starlings and pigeons.

Carriage of Class 1 and 2 Integrons in Acinetobacter baumannii and Pseudomonas aeruginosa Isolated from Clinical Specimens and a Novel Gene Cassette Array: blaOXA-11-cmlA7

The dissemination of antibiotic resistance genes between bacteria leads to serious problems in the treatment of infectious diseases. It has been shown that resistance genes can also be carried by the integrons. There are limited studies regarding the carriage of class 1 and 2 integrons in Acinetobacter baumannii and Pseudomonas aeruginosa clinical strains in Turkey. The aims of this study were to investigate the carriage rates of class 1 and class 2 integrons in A.baumannii and P.aeruginosa strains isolated from clinical samples in Abant Izzet Baysal University Hospital, and to characterize the antibiotic resistance gene cassettes in these integrons by sequence analyses. A total of 137 strains (77 A.baumannii and 60 P.aeruginosa) isolated from various clinical specimens (56% were sputum, 19% wound, 11% urine, 11% blood, 3% catheter), between March 2010-December 2012, were included in the study. The identification and antibiotic susceptibility tests of the isolates were performed by Vitek 2 Compact (bioMérieux, France) and BD Phoenix 100 (Becton Dickinson, USA) systems. The presence of integrons were screened by PCR method using specific primer pairs targeting class 1 (intI1) and 2 (intI2) integrase regions. All the samples that revealed integron amplification were subjected to DNA sequence analysis, both in the forms of cloned products and PCR amplicons. In the study, the highest susceptibility rates were found against colistin (96%) and tigecycline (78%) in A.baumannii, and against piperacillin/tazobactam (97%) and piperacillin (93%) in P.aeruginosa isolates. The highest resistance rate was determined for piperacillin/tazobactam (95%) in A.baumannii strains. The presence of intI1 gene was detected in 33% (26/77) of A.baumannii and 10% (6/60) of P.aeruginosa isolates. When variable regions in intI1 positive strains were amplified by PCR, eight (8/77, 10%) A.baumannii and three (3/60, 5%) P.aeruginosa strains were found to harbor antibiotic resistance gene cassettes. IntI2 gene was not detected in any of the isolates. Resistance to piperacillin/tazobactam, ceftazidime, cefepime, ceftriaxone and ampicillin/sulbactam was detected as the common resistance pattern in all integron-positive A.baumannii strains, whereas resistance to ceftazidime, gentamicin and ciprofloxacin was the common pattern in all integron-positive P.aeruginosa strains. DNA sequence analysis of variable regions of integrons indicated that two separate gene cassette arrays (aacC1-aadA1 and aac(3)-1) were carried by A.baumannii strains, and two types of gene cassette arrays (blaOXA-30-aadA1 and blaOXA-11-cmlA7) were carried by P.aeruginosa strains. To our best knowledge, this is the first report of the gene sequence of blaOXA-11-cmlA7 defined in an integron gene cassette of P.aeruginosa.

Quantitative and qualitative impact of hospital effluent on dissemination of the integron pool

There is increasing evidence that human activity, and especially the resulting effluent, has a major role in the dissemination of bacterial antibiotic-resistance determinants in the environment. Hospitals are the major antibiotic consumers and thus facilitate the spread of antibiotic resistance. Questions are increasingly being raised about the management of hospital effluents, but their involvement in antibiotic-resistance dissemination has never been assessed. Integrons are a paradigm of genetic transfer between the environmental resistome and both commensal and pathogenic bacteria. In order to assess the impact of hospital activities on antibiotic-resistance dissemination in the environment, we monitored integrons and their gene cassettes in hospital effluents, and their release in the environment. We found that bacterial communities present in a hospital effluent contained a high proportion of integrons. In terms of both their gene cassette diversity and gene cassette arrays, the urban effluent and municipal wastewater treatment plant (WWTP) influent were most similar, whereas the hospital effluent and recirculation sludge exhibited very specific patterns. We found that anthropogenic activities led to the release of abundant integrons and antibiotic-resistance gene cassettes, but we observed no specific impact of hospital activities on the receiving environment. Furthermore, although the WWTP did not reduce the normalized integron copy number, it reduced the diversity of gene cassette arrays contained in the raw wastewater, underlining the effect of the biological treatment on the anthropogenic integron pool arriving at the WWTP.

Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda, West Bengal, India

BackgroundIn this study a large random collection (n = 2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007–2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products.Methodology/Principal FindingsReplica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ2) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6′-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae.ConclusionsOligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.

Integron characterization and typing of Shiga toxin-producing Escherichia coli isolates in Belgium

The presence of integrons and the antibiotic susceptibility profiles of STEC strains isolated in Belgium were analysed. The collection contained 306 strains, of which 225 were human isolates and 81 originated from different food or animal sources. Integrons were detected by PCR in 7.5% of the tested isolates and all were class 1 integrons. The integron-positive strains all belonged to the human collection. By RFLP, five different types (A, B, C, D, E) were distinguished. The antibiotic-resistance gene cassettes were identified by sequencing representatives of the five different types. Two types of gene cassettes were found in different combinations, one encoding resistance to streptomycin/spectinomycin and the other encoding resistance to trimethoprim. One of the gene cassettes present was the rarely detected aadA23, which was now apparently for the first time reported in Western Europe. Susceptibility profiling of the strains for 11 antibiotics was done by standard disc diffusion assays. Among the 23 integron-positive strains, 17 different antibiotic susceptibility profiles were found. In the 283 integron-negative strains, 24 different antibiotic susceptibility profiles were observed. The majority of these strains were susceptible to all tested antibiotics (n=218, 77.0%). The integron-positive strains were significantly more resistant to eight of the eleven tested antibiotics compared to the integron-negative strains (P<0.05). PFGE profiles of integron-positive strains within selected serogroups did not cluster together.

Molecular characterization of theblaKPC-2gene in clinical isolates of carbapenem-resistantKlebsiella pneumoniaefrom the pediatric wards of a Chinese hospital

The present study was conducted to confirm the presence of Klebsiella pneumoniae carbapenemase (KPC)-producing K.pneumoniae associated with a nosocomial outbreak in a Chinese pediatric hospital. From July 2009 to January 2011, 124 nonduplicated K.pneumoniae isolates were collected from specimens from patients of pediatric units in the hospital. Twelve of the 124 isolates possessed the bla(KPC-2) gene and showed 7 different pulsed-field gel electrophoresis (PFGE) patterns. Meanwhile, 16S rRNA methylase, acc(6')-Ib-cr, and several types of β-lactamases were also produced by the majority of the KPC-producing isolates. Class 1 integron-encoded intI1 integrase gene was subsequently found in all strains, and amplification, sequencing, and comparison of DNA between 5' conserved segment and 3' conserved segment region showed the presence of several known antibiotic resistance gene cassettes of various sizes. The conjugation and plasmid-curing experiments indicated some KPC-2-encoding genes were transmissible. In addition, conjugal cotransfer of multidrug-resistant phenotypes with KPC-positive phenotypes was observed in KPC-producing strains. Restriction endonuclease analysis and DNA hybridization with a KPC-specific probe showed that the bla(KPC-2) gene was carried by plasmid DNA from K.pneumoniae of PFGE pattern B. The overall results indicate that the emergence and outbreak of KPC-producing K.pneumoniae in our pediatric wards occurred in conjunction with plasmids coharboring 16S rRNA methylase and extended-spectrum β-lactamases.

Diversity of Integron- and Culture-Associated Antibiotic Resistance Genes in Freshwater Floc

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.

RAC: Repository of Antibiotic resistance Cassettes

Antibiotic resistance in bacteria is often due to acquisition of resistance genes associated with different mobile genetic elements. In Gram-negative bacteria, many resistance genes are found as part of small mobile genetic elements called gene cassettes, generally found integrated into larger elements called integrons. Integrons carrying antibiotic resistance gene cassettes are often associated with mobile elements and here are designated ‘mobile resistance integrons’ (MRIs). More than one cassette can be inserted in the same integron to create arrays that contribute to the spread of multi-resistance. In many sequences in databases such as GenBank, only the genes within cassettes, rather than whole cassettes, are annotated and the same gene/cassette may be given different names in different entries, hampering analysis. We have developed the Repository of Antibiotic resistance Cassettes (RAC) website to provide an archive of gene cassettes that includes alternative gene names from multiple nomenclature systems and allows the community to contribute new cassettes. RAC also offers an additional function that allows users to submit sequences containing cassettes or arrays for annotation using the automatic annotation system Attacca. Attacca recognizes features (gene cassettes, integron regions) and identifies cassette arrays as patterns of features and can also distinguish minor cassette variants that may encode different resistance phenotypes (aacA4 cassettes and bla cassettes-encoding β-lactamases). Gaps in annotations are manually reviewed and those found to correspond to novel cassettes are assigned unique names. While there are other websites dedicated to integrons or antibiotic resistance genes, none includes a complete list of antibiotic resistance gene cassettes in MRI or offers consistent annotation and appropriate naming of all of these cassettes in submitted sequences. RAC thus provides a unique resource for researchers, which should reduce confusion and improve the quality of annotations of gene cassettes in integrons associated with antibiotic resistance.Database URL: http://www2.chi.unsw.edu.au/rac.

Regulation of the integrase and cassette promoters of the class 1 integron by nucleoid-associated proteins

The integrase IntI1 catalyses recombination of antibiotic-resistance gene cassettes in the integron, a widely found bacterial mobile element active in spreading antibiotic multi-resistance. We have previously shown that resistance cassette recombination rate and specificity depend on the amount of intracellular integrase. Here, we used in vivo and in vitro methods to examine convergent expression of the integrase promoter (P(int)) and of the cassette promoters (P(c) and P(2)) in the prototypical plasmid-borne class 1 integron, In2. Highly conserved P(int) has near consensus -10 and -35 hexamers for σ(70) RNA polymerase, but there are 11 naturally occurring arrangements of P(c) alone or combinations of the P(c)+P(2) cassette promoters (note that P(2) occurs with a 14 or 17 bp spacer). Using a bi-directional reporter vector, we found that P(int) is a strong promoter in vivo, but its expression is reduced by converging transcription from P(c) and P(2). In addition to cis-acting convergence control of integrase expression, the regulator site prediction program, prodoric 8.9, identified sites for global regulators FIS, LexA, IHF and H-NS in and near the integron promoters. In strains mutated in each global regulator, we found that: (1) FIS repressed integrase and cassette expression; (2) LexA repressed P(int) and P(2) with the 14 bp spacer version of P(2) and FIS was necessary for maximum LexA repression; (3) IHF activated P(int) when it faced the strong 17 bp spacer P(2) but did not elevate its expression versus LexA-repressed P(2) with the 14 bp spacer; and (4) H-NS repressed both P(int) and the 14 bp P(2) but activated the 17 bp P(2) cassette promoters. Mobility shift assays showed that FIS and IHF interact directly with the promoter regions and DNase I footprinting confirmed extensive protection by FIS of wild-type In2 integron promoter sequence. Thus, nucleoid-associated proteins, known to act directly in site-specific recombination, also control integron gene expression directly and possibly indirectly.

Characterization and horizontal transfer of class 1 integrons in Salmonella strains isolated from food products of animal origin

A total of 81 Salmonella isolates from retail meats and seafood in Hebei province, China, were assayed for the presence of and horizontal transfer of class 1 integrons. By the PCR screening for the integrons, class 1 integron was detected from strains in serotypes of Derby, Indiana, London and Choleraesuis, which were isolated from pork, chicken or seafood; however, two isolates contained the empty integron that lacked the resistance cassette, a potential hotspot for development of the multidrug resistance. In contrast, two other isolates had the antibiotic resistance gene cassettes within the class 1 integron, which were dfrA1-aadA1 and aadB-cmlA, respectively. The conjugation experiments demonstrated the plasmid-mediated transfer of the class 1 integrons. Furthermore, each of the integrons was transmitted to Streptococcus mutans via natural gene transformation. These findings suggest the possible transfer of class 1 integrons from foodborne pathogens to human residential bacteria via horizontal gene transfer.

Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species

Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.

Detection of class 1 and 2 integrons, extended-spectrum β-lactamases and qnr alleles in enterobacterial isolates from the digestive tract of Intensive Care Unit inpatients

In this study, we searched for extended-spectrum β-lactamases (ESBLs), class 1 and 2 integrons, and qnrA, qnrB and qnrS genes in 56 oxyimino-cephalosporin and/or ciprofloxacin-resistant enterobacterial isolates obtained from the gastrointestinal tract of patients admitted in an Intensive Care Unit in Uruguay. ESBLs were detected in 11 isolates (6 CTX-M-2, 3 CTX-M-9, 1 CTX-M-15 and 1 PER-2). qnr genes and integrons were detected in 5 and 24 isolates, respectively. Eight different antibiotic resistance gene cassettes were found within six different genetic arrangements. Two types of complex class 1 integrons carrying insertion sequence IS CR1 were found, one showing bla CTX-M-2–orf3 and the other qnrA1–ampR. Ten of the thirteen isolates carrying class 2 integrons presented the element IS 5 inserted between intI2 and dfrA1, whereas another class 2 integron lacked the internal stop codon usually present in intI2. This is the first report of the occurrence of qnrA, qnrB and bla CTX-M-9 in Uruguay. Dissemination of the different groups of CTX-M enzymes (i.e. groups 1, 2 and 9) appears to be a recent phenomenon in South America.

Analysis of clonally related environmental Vibrio cholerae O1 El Tor isolated before 1992 from Varanasi, India reveals origin of SXT‐ICEs belonging to O139 and O1 serogroups

In this study, we report the presence of SXT in environmental Vibrio cholerae O1 El Tor strains isolated before 1992 from Varanasi, India. All isolates, except one, were resistant to Tm, and/or Sul, Sm, Fr, Na and Am. None contained plasmids. PCR and DNA sequencing revealed the presence of SXT containing dfrA1 and/or sulII, strAB in six isolates and dfr18, sulII and strAB in five isolates. Three clinical V. cholerae O1 isolated during 1992 contained the antibiotic resistance gene cassette aadA1 in the class 1 integron. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence of the transferable nature of SXT and associated antibiotic resistance genes, and its integration into the prfC site. Results of phylogenetic analysis of the intSXT gene of clonally similar V. cholerae showed a clear difference between dfr18(+) and dfrA1(+) V. cholerae O1 isolates. This is the first report of the occurrence of SXT harbouring sulII, strAB, dfr18 and/or dfrA1 genes in environmental V. cholerae O1 isolated prior to 1992 from Varanasi, India, and suggests emergence of SXT(+) antibiotic-resistant V. cholerae O139 and O1 from an environmental V. cholerae progenitor by acquisition of SXT and antibiotic-resistant gene clusters.

Characterization of multidrug-resistant and extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains from Malaysian hospitals

The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae poses a serious antibiotic management problem as resistance genes are easily transferred from one organism to another. Fifty-one strains of K. pneumoniae isolated from sporadic cases in various hospitals throughout Malaysia were analysed by antimicrobial susceptibility testing, PCR detection of ESBL-encoding genes and DNA fingerprinting. Although 27 of the 51 K. pneumoniae strains were MDR (i.e. resistant to three or more classes of antibiotics), the majority of the strains (98 %) were sensitive to imipenem. PCR detection using ESBL gene-specific primers showed that 46 of the K. pneumoniae strains harboured bla(SHV), 19 harboured bla(CTX-M), 5 harboured bla(OXA-1) and 4 harboured bla(TEM-1). Class 1 integron-encoded intI1 integrase was detected in 21 of the 51 K. pneumoniae strains and amplification of the integron 5'CS region showed the presence of several known antibiotic resistance gene cassettes of various sizes. Results of conjugation and transformation experiments indicated that some of the ESBL-encoding genes (i.e. bla(SHV), bla(CTX-M) and bla(TEM-1)) were transmissible and were likely plasmid-encoded. DNA fingerprinting using PFGE and PCR-based methods indicated that the 51 K. pneumoniae strains were genetically diverse and heterogeneous.

Context-driven discovery of gene cassettes in mobile integrons using a computational grammar

BackgroundGene discovery algorithms typically examine sequence data for low level patterns. A novel method to computationally discover higher order DNA structures is presented, using a context sensitive grammar. The algorithm was applied to the discovery of gene cassettes associated with integrons. The discovery and annotation of antibiotic resistance genes in such cassettes is essential for effective monitoring of antibiotic resistance patterns and formulation of public health antibiotic prescription policies.ResultsWe discovered two new putative gene cassettes using the method, from 276 integron features and 978 GenBank sequences. The system achieved κ = 0.972 annotation agreement with an expert gold standard of 300 sequences. In rediscovery experiments, we deleted 789,196 cassette instances over 2030 experiments and correctly relabelled 85.6% (α ≥ 95%, E ≤ 1%, mean sensitivity = 0.86, specificity = 1, F-score = 0.93), with no false positives.Error analysis demonstrated that for 72,338 missed deletions, two adjacent deleted cassettes were labeled as a single cassette, increasing performance to 94.8% (mean sensitivity = 0.92, specificity = 1, F-score = 0.96).ConclusionUsing grammars we were able to represent heuristic background knowledge about large and complex structures in DNA. Importantly, we were also able to use the context embedded in the model to discover new putative antibiotic resistance gene cassettes. The method is complementary to existing automatic annotation systems which operate at the sequence level.

Highly Variable Patterns of Antimicrobial Resistance in CommensalEscherichia coliIsolates from Pigs, Sympatric Rodents, and Flies

Antimicrobial-resistant Escherichia coli strains from pigs, sympatric rodents, and flies from two large farms in the Czech Republic with different antibiotic exposure histories were characterized based on antimicrobial resistance genes, integrons, and macrorestriction DNA profiles. Isolates of E. coli were tested for susceptibility to 12 antimicrobial agents according to the standard disk diffusion method. In resistant isolates, polymerase chain reaction was used to detect antibiotic resistance genes, integrase genes, and gene cassettes. Pulsed-field gel electrophoresis (PFGE) was used for molecular subtyping of E. coli. In farm A (long-term use of amoxicillin only), 75% (n = 198), 65% (n = 49), 11% (n = 139), and 82% (n = 177) of E. coli isolates from piglets, sows, sympatric rodents, and flies, respectively, were antibiotic resistant. In farm B (various antibiotics commonly used), 53% (n = 154), 69% (n = 98), and 54% (n = 74) of E. coli isolates from piglets, sows, and sympatric rodents, respectively, were antibiotic resistant. In both farms, the highest resistance prevalence was to tetracycline, and resistance patterns of isolates were greatly variable. Isolates with the same resistance phenotype, genes, and PFGE profile were found in pigs and flies. Isolates from rodents showed unique PFGE profiles. Close contact of sympatric rodents and flies with pigs or their products was associated with colonization of rodents and flies with resistant bacteria or transfer of resistance genes found in pig intestinal flora.

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

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 beta-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 beta-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.

Gene cassettes encoding resistance to quaternary ammonium compounds: a role in the origin of clinical class 1 integrons?

DNA sequencing, phylogenetic and mapping studies suggest that the class 1 integron found in pathogens arose when one member of the diverse family of environmental class 1 integrons became embedded into a Tn402 transposon. However, the timing of this event and the selective forces that first fixed the newly formed element in a bacterial lineage are still unknown. Biocides have a longer use in clinical practice than antibiotics, and a qac (quaternary ammonium compound) resistance gene, or remnant thereof, is a normal feature of class 1 integrons recovered from clinical isolates. Consequently, it is possible that the initial selective advantage was conferred by resistance to biocides, mediated by qac. Here, we show that diverse qac gene cassettes are a dominant feature of cassette arrays from environmental class 1 integrons, and that they occur in the absence of any antibiotic resistance gene cassettes. They are present in arrays that are dynamic, acquiring and rearranging gene cassettes within the arrays. The abundance of qac gene cassettes makes them a likely candidate for participation in the original insertion into Tn402, and as a source of a readily selectable phenotype. More broadly, the increasing use of qac and other biocides at the present time seems likely to promote the fixation of further novel genetic elements, with unpredictable and potentially adverse consequences for human health and agriculture.

Seagulls of the Berlengas Natural Reserve of Portugal as Carriers of Fecal Escherichia coli Harboring CTX-M and TEM Extended-Spectrum Beta-Lactamases

Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.

The IL-8 Protease SpyCEP/ScpC of Group A Streptococcus Promotes Resistance to Neutrophil Killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.