Experiences in fosfomycin susceptibility testing and resistance mechanism determination in Escherichia coli from urinary tract infections in the UK.

PURPOSE: With an increase in the numbers of bacterial isolates resistant to first-line antibiotics, there has been a revival in the use of older drugs including fosfomycin with novel mechanisms of action. We aimed to investigate the prevalence and genotypic nature of fosfomycin resistance in Escherichia coli from urinary tract infections (UTIs) using the various methods available in the clinical microbiology laboratory. METHODOLOGY: In total, 1000 culture-positive urine samples were assessed for the presence of E. coli and fosfomycin susceptibility was determined using the MAST Uri system, microbroth dilution, agar dilution and E-test strips.Results/Key findings. Initial investigation using breakpoint susceptibility testing on the MAST Uri system identified 62 of 657 (9.5 %) E. coli isolates as fosfomycin-resistant (MIC≥32 µg ml-1). However, on further testing, a lower rate of eight of the 62 (1.3 %) were robustly confirmed to be resistant using microbroth dilution, agar dilution and E-test strips. These true resistant isolates belonged to diverse E. coli multi-locus sequence types and each had a unique set of chromosomal alterations in genes associated with fosfomycin resistance. Fosfomycin-resistant isolates were not multiply drug resistant and did not carry plasmidic fosfomycin resistance genes. Therefore, the use of fosfomycin may be unlikely to drive selection of a particular clone or movement of transferrable resistance genes. CONCLUSION: Fosfomycin remains a viable option for the treatment of E. coli in uncomplicated UTIs; different susceptibility testing platforms can give very different results regarding the prevalence of fosfomycin resistance, with false positives being a potential problem that may unnecessarily limit the use of this agent.

Cryptic silver resistance is prevalent and readily activated in certain Gram-negative pathogens.

OBJECTIVES: To assess the prevalence of cryptic silver (Ag+) resistance amongst clinical isolates of Gram-negative bacteria, and to examine how overt Ag+ resistance becomes activated in such strains. METHODS: Established methods were used to determine the susceptibility of 444 recent clinical isolates to Ag+, and to evaluate the potential for overt Ag+ resistance to emerge in susceptible isolates by spontaneous mutation. The genetic basis for Ag+ resistance was investigated using PCR amplification and DNA sequencing. RESULTS: None of the isolates tested displayed overt Ag+ resistance. However, upon silver challenge, high-level Ag+ resistance (silver nitrate MIC >128 mg/L) was selected at high frequency (10-7 to 10-8) in 76% of isolates of Enterobacter spp., ∼58% of isolates of Klebsiella spp. and ∼0.7% of isolates of Escherichia coli. All strains in which Ag+ resistance could be selected harboured the sil operon, with resistance apparently resulting from activation of this system as a consequence of single missense mutations in silS. By contrast, Ag+ resistance was not selected in isolates lacking sil, which included all tested representatives of Pseudomonas aeruginosa, Acinetobacter spp., Citrobacter spp. and Proteus spp. CONCLUSIONS: Whilst overt Ag+ resistance in Gram-negative pathogens is uncommon, cryptic Ag+ resistance pertaining to the sil operon is prevalent and readily activated in particular genera (Enterobacter and Klebsiella).

Functional genomics to identify the factors contributing to successful persistence and global spread of an antibiotic resistance plasmid.

BACKGROUND: The spread of bacterial plasmids is an increasing global problem contributing to the widespread dissemination of antibiotic resistance genes including ß-lactamases. Our understanding of the details of the biological mechanisms by which these natural plasmids are able to persist in bacterial populations and are able to establish themselves in new hosts via conjugative transfer is very poor. We recently identified and sequenced a globally successful plasmid, pCT, conferring ß-lactam resistance. RESULTS: Here, we investigated six plasmid encoded factors (tra and pil loci; rci shufflon recombinase, a putative sigma factor, a putative parB partitioning gene and a pndACB toxin-antitoxin system) hypothesised to contribute to the 'evolutionary success' of plasmid pCT. Using a functional genomics approach, the role of these loci was investigated by systematically inactivating each region and examining the impact on plasmid persistence, conjugation and bacterial host biology. While the tra locus was found to be essential for all pCT conjugative transfer, the second conjugation (pil) locus was found to increase conjugation frequencies in liquid media to particular bacterial host recipients (determined in part by the rci shufflon recombinase). Inactivation of the pCT pndACB system and parB did not reduce the stability of this plasmid. CONCLUSIONS: Our findings suggest the success of pCT may be due to a combination of factors including plasmid stability within a range of bacterial hosts, a lack of a fitness burden and efficient transfer rates to new bacterial hosts rather than the presence of a particular gene or phenotype transferred to the host. The methodology used in our study could be applied to other 'successful' globally distributed plasmids to discover the role of currently unknown plasmid backbone genes or to investigate other factors which allow these elements to persist and spread.

Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in the bovine fecal metagenome.

The study objective was to determine the effects of two treatment regimens on quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The two regimens were ceftiofur crystalline-free acid (CCFA) administered to either one or all steers within a pen and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. A 26-day randomized controlled field trial was conducted on 176 steers. Real-time PCR was used to quantify bla(CMY-2), bla(CTX-M), tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community DNA. A significant increase in ceftiofur resistance and a decrease in tetracycline resistance elements were observed among the treatment groups in which all steers received CCFA treatment, expressed as gene copies/gram of feces. Subsequent chlortetracycline administration led to rapid expansion of both ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest that chlortetracycline is contraindicated when attempting to avoid expansion of resistance to critically important third-generation cephalosporins.

Effects of ceftiofur and chlortetracycline treatment strategies on antimicrobial susceptibility and on tet(A), tet(B), and bla CMY-2 resistance genes among E. coli isolated from the feces of feedlot cattle.

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P<0.05). The four treatment regimens also influenced the phenotypic multidrug resistance count of NTS E. coli populations. Chlortetracycline treatment alone was associated with an increased probability of selecting isolates that harbored tet(B) versus tet(A) (P<0.05); meanwhile, there was an inverse association between finding tet(A) versus tet(B) genes for any given regimen (P<0.05). The presence of a tet(A) gene was associated with an isolate exhibiting reduced phenotypic susceptibility to a higher median number of antimicrobials (n = 289, median = 6; 95% CI = 4-8) compared with the tet(B) gene (n = 208, median = 3; 95% CI = 3-4). Results indicate that CTC can exacerbate ceftiofur resistance following CCFA therapy and therefore should be avoided, especially when considering their use in sequence. Further studies are required to establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together.

Persistence of transferable extended-spectrum-ß-lactamase resistance in the absence of antibiotic pressure.

The treatment of infections caused by antibiotic-resistant bacteria is one of the great challenges faced by clinicians in the 21st century. Antibiotic resistance genes are often transferred between bacteria by mobile genetic vectors called plasmids. It is commonly believed that removal of antibiotic pressure will reduce the numbers of antibiotic-resistant bacteria due to the perception that carriage of resistance imposes a fitness cost on the bacterium. This study investigated the ability of the plasmid pCT, a globally distributed plasmid that carries an extended-spectrum-ß-lactamase (ESBL) resistance gene (bla(CTX-M-14)), to persist and disseminate in the absence of antibiotic pressure. We investigated key attributes in plasmid success, including conjugation frequencies, bacterial-host growth rates, ability to cause infection, and impact on the fitness of host strains. We also determined the contribution of the bla(CTX-M-14) gene itself to the biology of the plasmid and host bacterium. Carriage of pCT was found to impose no detectable fitness cost on various bacterial hosts. An absence of antibiotic pressure and inactivation of the antibiotic resistance gene also had no effect on plasmid persistence, conjugation frequency, or bacterial-host biology. In conclusion, plasmids such as pCT have evolved to impose little impact on host strains. Therefore, the persistence of antibiotic resistance genes and their vectors is to be expected in the absence of antibiotic selective pressure regardless of antibiotic stewardship. Other means to reduce plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes they carry.

Dissemination of pCT-like IncK plasmids harboring CTX-M-14 extended-spectrum ß-lactamase among clinical Escherichia coli isolates in the United Kingdom.

IncK plasmids encoding CTX-M-14 extended-spectrum ß-lactamase (ESBL) and highly related to plasmid pCT were detected in 13 of 67 (19%) human clinical isolates of Escherichia coli with a group 9 CTX-M-type ESBL from the United Kingdom and in 2 quality assurance isolates. None of these E. coli strains was related to the cattle strain from which pCT was originally characterized.

Complete sequence and molecular epidemiology of IncK epidemic plasmid encoding blaCTX-M-14.

Antimicrobial drug resistance is a global challenge for the 21st century with the emergence of resistant bacterial strains worldwide. Transferable resistance to ß-lactam antimicrobial drugs, mediated by production of extended-spectrum ß-lactamases (ESBLs), is of particular concern. In 2004, an ESBL-carrying IncK plasmid (pCT) was isolated from cattle in the United Kingdom. The sequence was a 93,629-bp plasmid encoding a single antimicrobial drug resistance gene, blaCTX-M-14. From this information, PCRs identifying novel features of pCT were designed and applied to isolates from several countries, showing that the plasmid has disseminated worldwide in bacteria from humans and animals. Complete DNA sequences can be used as a platform to develop rapid epidemiologic tools to identify and trace the spread of plasmids in clinically relevant pathogens, thus facilitating a better understanding of their distribution and ability to transfer between bacteria of humans and animals.

RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium.

The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.