deep-sea microflora. insertions in the vicinity of these genes have been shown to increase the strength of their promoters and increase resistance. because of these characteristics, a wide geographic dissemination of oxa carbapenemase resistance in particular has occurred. the facilitated spread of carbapenem resistance appears to have multiple origins and repeated introduction into the uk of bacteria with the blaoxa-48 gene via horizontal transfer of similar plasmids to poxa-48a. a recent study in the uk examined 26 isolates of enterobacteriaceae consisting of a diverse set of sequence types (st) of k. pneumoniae, e. coli, and enterobacter cloacae producing oxa-48-like carbapenemases. their findings included: 25 of the 26 strains had the blaoxa-48 gene. 21 of these isolates had resistance plasmids that could be transferred by conjugation; 20 of these transformants had the three functional genes, repa, trau, and para found in poxa-48a. in st38 e. coli, no oxa-48 transconjugants were found and it only had the para gene. the indian strain of k. pneumonia had an oxa-181-encoding plasmid (which had higher resistance to carbapenem) and also could not be transferred by conjugation and had none of the three functional genes found in poxa-48a. gram-negative bacteria outer membrane vesicles (omvs), that can transfer dna between bacterial cells, are produced by metabolically active bacterial cells, and the omvs are not the result of cell lysis or cell death. pathogenic strains can produce about 10-25 times more vesicles than a nonpathogenic strain making this highly relevant to carbapenem resistance transfer. omvs protect plasmids from being digested extracellularly by nucleases that may be found in the environment, thus favoring horizontal gene transfer. laboratory analysis agar plate method variations are seen in the media used for inoculation. many studies use media with 1 to 2 mg/l of imipenem. however, bacteria that produce oxa-48 or oxa-181 result in low-level resistance, which cannot be detected efficiently due to the high concentration. therefore, more recent screening media use broth containing 0.5–1 mg/l imipenem or 0.5 mg/l ertapenem. the downsides to this approach include the delay of results from the inoculation and the inability to identify the type of carbapenemase. disc diffusion method the disc diffusion method can be used by hospital laboratories to screen for cre. in this technique, antibiotic discs are placed onto plates of mueller hinton agar that have already been inoculated with the sample strain. the plates are then incubated overnight at 37 °c. following incubation, the zones of inhibition surrounding the various antibiotic discs are measured and compared with clinical and laboratory standard institute guidelines. identification of kpcs, mbls and oxas can be achieved by demonstrating synergistic inhibition with phenyl boronic acid, edta or neither, respectively. in a thailand-based study of cre in hospital settings, carbapenem resistance was defined as any strain that shows resistance to at least one of three carbapenem antibiotics tested. pcr method pcr-based screening methodologies are in the process of development for a number of genes responsible for resistance. they speed up detection and can readily differentiate between carbapenamase genes-sometimes in a multiplex format. costs of pcr testing are decreasing and reliability of molecular based tests relates more to gene presence and gene expression for production of the relevant carbapenamase. nested arbitrary pcr (arb-pcr) was used during a 2007 cre outbreak at the university of virginia medical center to identify the specific bla kpc plasmid involved in the transmission of the infection, and researchers suggest that arb-pcr may also be used to identify other methods of cre spread. maldi-tof ms another study used matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof ms) to determine resistance patterns in bacteria from freshly positive blood cultures. maldi-tof ms can detect changes in mass to charge ratios. cabapenem-resistant bacteria often employ β-lactamases, which physically disrupt the structure of β-lactam antibiotics. since this causes a change in the mass of the antibiotic, resistant bacteria are detectable by maldi-tof ms. accepted clinical tests often require an overnight incubation before reading the result, but maldi-tof ms can return results in just 4–5 hours. maldi-tof cannot detect resistant bacteria, which do not physically disrupt the β-lactam antibiotic, i.e. where no mass change occurs. therefore, the method serves best as a first screen for patients admitted to the hospital, but should be followed with secondary testing. prevention researchers found environmental reservoirs of cre bacteria in icu sinks and drains. despite multiple attempts to sterilize these sinks and drains, using detergents and steam, the hospital staff was unsuccessful in getting rid of the cre. due to the bacterial resistance to cleaning measures, staff should take extreme precaution in maintaining sterile environments in hospitals not yet infected with the cre-resistant bacteria. a major means of transmission is through sinks, so staff should take extra precaution in maintaining sterile conditions. hospitals could reduce transmission by creating sinks with designs that could reduce backsplash. another method to reduce transmission from sink to sink is to have sink brushes in each room that would be for cleaning that individual sink alone. hospital staff should be trained to never dispose of clinical waste down the sinks in patient rooms. a hospital in melbourne, australia, implemented similar strategies as these to reduce transmission and prevent further infection of more icu patients. armed with the knowledge of their status as cre transmission sites, hospitals must take special care to monitor cre outbreaks within their wards. efficient and accurate detection of cre is the first step. enterobacteriaceae are most commonly found in the intestinal flora. using stool and rectal swabs are, thus, the most reliable methods for testing resistance. no billing code exists for cre under medicare or medicaid, making it difficult to track on a national level in the u.s. another challenge facing efforts to control transmission is the fact that although long-term care facilities have been heavily indicated as the primary centers for incidence, amplification, and spread of cre, studies that have controlled for this transmission have still found cre spreading in other affiliated hospitals, indicating that long-term acute-care facilities are likely not the sole culprit in the spread of cre and other multidrug-resistant organisms. one method found effective is to screen and isolate incoming patients from other facilities, and renew focus on hand washing. no new drugs for the bacteria are in development and the bacteria's rapid adaptation to new drugs makes investment in their development unprofitable, as the new drug would quickly become useless. studies have found that cre incidence and prevalence can be reduced by applying targeted interventions including increased hygiene measures and equipment sterilization, even in populations where the prevalence of infection exceeds 50% of patients. however, additional environmental cleaning to control transmission has not been verified by controlled trials. the involvement of local and national public health authorities will likely be critical to ensure broader and more sustainable implementation of these measures. prevention is a top priority for reducing person-to-person transmission of cre. this is especially true because limited treatment options are available to use after carbapenem resistance develops. most current research calls for a coordinated, multifaceted approach to infection prevention and containment, and the centers for disease control and prevention have issued preliminary guidelines for the control of cre transmission. experts advocate for a proactive approach, based on the belief that it will be most cost-effective to combat the problem before it is established. however, when immediate financial and personnel resources are limited, healthcare administrators may be forced to respond reactively, aiming to reduce any further transmission. although a consensus exists for the need of prevention protocols, infection control practices often vary among hospitals, even within close geographic area. in a survey of 15 hospitals within the toronto area, many hospitals employed varying combinations of basic infection control practices. eight different practices were observed among the 15 hospitals, some of which included in the most recent publication of guidelines from the public health agency of canada. some of these recommendations include laboratory testing, active surveillance, screening (rectal swab, urine culture), hand hygiene, personal protective equipment, environmental cleaning, laundry waste management, and isolation with dedicated equipment and nursing staff. however, only five hospitals had written policies describing how to respond to an outbreak. many public health initiatives are moving towards a more standardized approach at multiple levels: among local facilities (especially long-term and acute care), regional hospitals, national institutions, and global practices. a standardized approach of prevention may help to more effectively reduce the emergence of cre. an infection control plan was implemented at the kaplan medical center in israel to control a hospital outbreak of carbapenem-resistant k. pneumoniae. the comprehensive plan included guidelines for cohorting patients in separate locations, cleaning with 1,000 ppm hypochlorite, screening for isolates from rectal swabs, and distribution of educational instruction sheets, lectures for all medical staff, and training. the hospital also implemented an automated computer system that updated patient charts when new cases were reported, if patients were carriers, and what precautions to take when dealing with such patients. this plan was evaluated in a quasiexperimental study through the incidence of clinical cases, the rate of cross-infection, and the rate of screening for carriage in admitted patients with increased risk of carriage. the study had a 16-fold decrease in the incidence of resistant k. pneumoniae, which was sustained for 30 months. the plan can provide a model for other hospitals to contain outbreaks of carbapenem-resistant bacteria. a reduction in the use of unnecessary invasive devices, including urinary catheters, could help reduce cre transmission. several methods have been tested for their effectiveness at improving thorough intensive-care unit environmental hygiene. a study conducted in 2010 across 3532 high risk environmental surfaces in 260 intensive care unit rooms in 27 acute-care hospitals (icus) assessed the consistency at which these surfaces met base line cleaning standards. only 49.5% of the high-risk object surfaces were found to meet this baseline criterion. the least-cleaned objects were bathroom light switches, room door knobs, and bed pan cleaners. significant improvements in icu room cleaning was achieved through a structured approach that incorporated a simple, highly objective surface targeting method and repeated performance feedback to environmental surface personnel. specific methods included implementing an objective evaluation process, environmental surfaces staff education, programmatic feedback, and continuous training to minimize the spread of hospital-associated infections. the authors noted an improvement in the thoroughness of cleaning at 71% from baseline for the entire group of hospitals involved. treatments fosfomycin several antimicrobial drugs have been tested for the effective treatment of cre. fosfomycin is an antimicrobial agent that acts to inhibit udp-n-acetylglucosamine enolpyruvyl transferase (mura) which catalyzes one of the early steps of bacterial cell wall synthesis, and is effective against gram-negative and -positive aerobic bacteria, such as cre. a meta-analysis of 17 studies investigating the clinical effectiveness of fosfomycin in four multidrug-resistant strains of enterobacteriaceae found 11 which reported that over 90% of bacterial isolates were susceptible to fosfomycin. the elevated level of antimicrobial activity by fosfomycin can be attributed to the fact that resistance to this antibiotic in enterobacteriaceae is chromosomally encoded and not plasmid-mediated. this causes a decreased capacity for survival in the bacteria. bacteria that are naturally resistant to fosfomycin are less robust and less pathogenic. tigecycline tigecycline, a member of the glycylcyclines antibiotics, has proven to be an effective therapy against enterobacteriaceae that typically display tetracycline resistance, because tigecycline has a higher binding affinity with ribosomal sites than tetracycline has. tigecycline is capable of killing almost all of the esbls and multidrug-resistant (mdr) e. coli isolates and the large majority of esbl and mdr isolates of klebsiella species. a 2008 review of 42 studies of in vitro susceptibility of bacteria to tigecycline showed that mdr k. pneumoniae and e. coli, including those that were carbapenem resistant, were susceptible more than 90% of the time. a limited number of patients have been treated with tigecycline, but the fda has approved it in certain cases with synergies of other drugs. the limited number of patients indicates that more trials are needed to determine the overall clinical effectiveness. although tigecycline is one of the first lines of defense against carbapenemase-producing isolates, negative clinical outcomes with tigecycline have occurred. both urinary tract and primary blood infections can make tigecycline ineffective, because it has limited penetration and rapid tissue diffusion after being intravenously infused, respectively. other antibiotics alternatives to fosfomycin include nitrofurantoin, pivmecillinam, and co-amoxiclav in oral