Metallo-beta-lactamase-producing Escherichia coli in the sewage of Mexico City: where do they come from?

While monitoring the presence of antibiotic resistance in municipal wastewater bacteria from Mexico City, five Escherichia coli isolates were found to be resistant to carbapenems, antibiotics of "last resort" used mostly in hospitals. Further analysis revealed that these carbapenem-resistant isolates carried the gene encoding a metallo-beta-lactamase, NDM-5. The gene was found to be beared by a large, ∼145 kb conjugative plasmid, which also carries putative genes encoding resistance to sulfonamides, trimethoprim, tetracycline, ciprofloxacin, and chloramphenicol (although no phenotypic chloramphenicol resistance was detected) and quaternary-ammonium compounds. The plasmid also carried gene mobility determinants, such as integron integrase and two transposases. In addition to the direct public health threat posed by the presence of such multi-resistant organisms in wastewater released into the environment and used for crop irrigation; it is particularly concerning that carbapenem-resistant E. coli is rather rare in Mexican hospitals (<1%), but was found in small, 100 mL samples of municipal wastewater. This suggests that these organisms are under-reported by clinical microbiology laboratories, underlining the usefulness of wastewater monitoring, or that there is an unknown source of such carbapenem-resistant organisms that are being dumped into the wastewater. The source of these bacteria must be assessed and controlled to prevent further spread of this multi-resistance plasmid among other environmental and clinical microorganisms.

Characterization of Escherichia coli Isolates from an Urban Lake Receiving Water from a Wastewater Treatment Plant in Mexico City: Fecal Pollution and Antibiotic Resistance.

The presence of enteric bacteria in water bodies is a cause of public health concerns, either by directly causing water- and food-borne diseases, or acting as reservoirs for antibiotic resistance determinants. Water is used for crop irrigation; and sediments and aquatic plants are used as fertilizing supplements and soil conditioners. In this work, the bacterial load of several micro-environments of the urban lake of Xochimilco, in Mexico City, was characterized. We found a differential distribution of enteric bacteria between the water column, sediment, and the rhizoplane of aquatic plants, with human fecal bacteria concentrating in the sediment, pointing to the need to assess such bacterial load for each micro-environment, for regulatory agricultural purposes, instead of only the one of the water, as is currently done. Resistance to tetracycline, ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole was common among Escherichia coli isolates, but was also differentially distributed, being again higher in sediment isolates. A distinct distribution of chloramphenicol minimum inhibitory concentrations (MIC) among these isolates suggests the presence of a local selective pressure favoring lower MICs than those of isolates from treated water. Fecal bacteria of human origin, living in water bodies along with their antibiotic resistance genes, could be much more common than typically considered, and pose a higher health risk, if assessments are only made on the water column of such bodies.

Non-Canonical Aspects of Antibiotics and Antibiotic Resistance.

The understanding of antibiotic resistance, one of the major health threats of our time, is mostly based on dated and incomplete notions, especially in clinical contexts. The "canonical" mechanisms of action and pharmacodynamics of antibiotics, as well as the methods used to assess their activity upon bacteria, have not changed in decades; the same applies to the definition, acquisition, selective pressures, and drivers of resistance. As a consequence, the strategies to improve antibiotic usage and overcome resistance have ultimately failed. This review gathers most of the "non-canonical" notions on antibiotics and resistance: from the alternative mechanisms of action of antibiotics and the limitations of susceptibility testing to the wide variety of selective pressures, lateral gene transfer mechanisms, ubiquity, and societal factors maintaining resistance. Only by having a "big picture" view of the problem can adequate strategies to harness resistance be devised. These strategies must be global, addressing the many aspects that drive the increasing prevalence of resistant bacteria aside from the clinical use of antibiotics.

Nitrofurantoin, phenazopyridine, and the superoxide-response regulon soxRS of Escherichia coli.

Nitrofurantoin and phenazopyridine are two drugs commonly used against urinary tract infections. Both compounds exert oxidative damage in patients deficient in glucose-6-phosphate dehydrogenase. This study was done to assess the interactions of these drugs with the soxRS regulon of Escherichia coli, a superoxide-defense system (that includes a nitroreductase that yields the active metabolite of nitrofurantoin) involved in antibiotic multi-resistance. The effects of either nitrofurantoin or phenazopyridine, upon strains with different soxRS genotypes, were measured as minimum inhibitory concentrations (MICs) and growth curves. Also, the ability of these drugs to induce the expression of a soxS'::lacZ gene fusion was assessed. The effect of antibiotics in the presence of phenazopyridine, paraquat (a known soxRS inducer), or an efflux inhibitor, was measured using the disk diffusion method. A strain constitutively expressing the soxRS regulon was slightly more susceptible to nitrofurantoin, and more resistant to phenazopyridine, compared to wild-type and soxRS-deleted strains, during early treatment, but 24-h MICs were the same (8 mg/l nitrofurantoin, 1,000 mg/l phenazopyridine) for all strains. Both compounds were capable of inducing the expression of a soxS'::lacZ fusion, but less than paraquat. Subinhibitory concentrations of phenazopyridine increased the antimicrobial effect of ampicillin, chloramphenicol, tetracycline, and nitrofurantoin. The induction or constitutive expression of the soxRS regulon seems to be a disadvantage for E. coli during nitrofurantoin exposure; but might be an advantage during phenazopyridine exposure, indicating that the latter compound could act as a selective pressure for mutations related to virulence and antibiotic multi-resistance.

Macrolides at Clinically-Relevant Concentrations May Induce Biofilm Formation in Macrolide-Resistant Staphylococcus aureus.

Macrolides inhibit biofilm formation in several Gram-negative, intrinsically-resistant bacterial species. However, the effect of macrolides upon biofilm formation by susceptible Gram-positive bacteria has been much less explored as such concentrations also inhibit cell growth. To circumvent this problem, the effect of macrolides (erythromycin, clarithromycin and azithromycin) at 0.5-2 µg/mL, upon biofilm formation, was explored on macrolide-resistant Staphylococcus aureus isolates, using the crystal violet assay with 96-well plates. Early (4 h) biofilm formation by strains having constitutive target-modification resistance was consistently induced by all macrolides but not in azithromycin-treated cells in longer (8 and 12 h) incubation. In inducible-resistance isolates, early biofilm formation was enhanced by some macrolide treatments, compared to similar cell growth in the absence of antibiotics; but the typical decay of biofilms at longer incubation appeared prematurely in macrolide-treated cultures. Biofilm formation in an efflux-mediated resistant isolate was not affected by macrolides. These results indicate that macrolides induce the formation of biofilm by resistant S. aureus isolates, especially during the early stages. This suggests that the empirical use of macrolides against infections caused by resistant S. aureus strains could not only result in clinical failure but even in the enhancement of biofilms, making further treatment difficult.

Ascorbate and Antibiotics, at Concentrations Attainable in Urine, Can Inhibit the Growth of Resistant Strains of Escherichia coli Cultured in Synthetic Human Urine.

There are conflicting reports on the antibacterial activity of ascorbate; all at concentrations much higher than the typical in human plasma, but that can be reached in urine. The effect of 10 mM ascorbate (in itself not inhibitory) along with antibiotics, was tested both in Mueller-Hinton broth (MHb) and in synthetic human urine (SHU), against resistant isolates of Escherichia coli from lower urinary infections. The activity of nitrofurantoin and sulfamethoxazole was higher in SHU than in MHb; minimal inhibitory concentrations (MICs) in SHU with ascorbate were below typical urinary concentrations. For other antibiotics, MICs were the same in MHb vs. SHU, with no effect of ascorbate in MHb; but in SHU with ascorbate, MICs of ciprofloxacin and gentamicin also went below reported urinary concentrations, with a lesser effect with norfloxacin and trimethoprim, and none with ampicillin. The effect of ascorbate was independent of oxygen and not related to the susceptibility of each strain to oxidative stress. Ascorbate oxidizes during incubation in SHU, and bacterial growth partially prevented oxidation. These results suggest that 10 mM ascorbate can enhance the inhibitory activity of antibiotics upon resistant strains in urine. Clinical experimentation with ascorbate-antibiotic combinations against urinary infections caused by resistant bacteria is warranted.

Phage Therapies: Lessons (Not) Learned from the "Antibiotic Era".

The use of phages as therapeutic or prophylactic approaches is gaining increased interest amid the growing menace of antibiotic resistance. Phages, along with other new anti-infective strategies, are certainly welcome as much needed additions to the medicinal arsenal. However, we can easily make with phages the same mistakes we made with antibiotics, which caused the current resistance crisis. The oversimplification of the ecological role of antibiotics, neglecting ancient resistance and the role of horizontal gene transfer; the active search for wide spectrum, and the massive agricultural abuse; and, most importantly, the financial greed behind the development and use of antibiotics; these are all trends that are now visible in phage research. Should we bring phages to the same track that wasted antibiotics, we could be looking at a "postphage era" in our near future.

Myths and Misconceptions around Antibiotic Resistance: Time to Get Rid of Them.

The antibiotic resistance arena is fraught with myths and misconceptions, leading to wrong strategies to combat it. It is crucial to identify them, discuss them in light of current evidence, and dispel those that are unequivocally wrong. This article proposes some concepts that may qualify as misconceptions around antibiotic resistance: the susceptible-resistant dichotomy; that incomplete antibiotic courses cause resistance; that resistance "emerges" in patients and hospitals; that antibiotics are mostly abused clinically; that resistance is higher in countries that use more antibiotics; that reducing antibiotic usage would reduce resistance; that financial incentives would "jumpstart" research and development of antibiotics; that generic and "original" antibiotics are the same; and that new anti-infective therapies are just around the corner. While some of these issues are still controversial, it is important to recognize their controversial status, instead of repeating them in specialized literature and lectures and, especially, in the planning of strategies to cope with resistance.

Antibiotic usage and resistance in Mexico: an update after a decade of change.

Ten years ago, a review on the status of resistance in Mexico was bleak: with antibiotics freely sold over the counter and poor regulation of generic drugs, among other conditions, resistance among relevant pathogens often ranked top, either among Latin American countries, or even worldwide. Since then, there have been some regulatory changes, along a decline in antibiotics usage: antibiotics are (supposedly) no longer sold without prescription, generic drugs (supposedly) have to demonstrate bioequivalence, and antibiotic usage has drop, from about 13 defined daily doses per 1,000 inhabitants per day, to 7. While these changes may sound encouraging, an analysis show that regulatory changes are incomplete at best, and usage decline may be the consequence of factors such as growing poverty. The assessment of resistance continues to be haphazard, without an organized network of laboratories providing a coherent picture. However, judging from a few nationwide reports, it appears that resistance among some nosocomial pathogens (MRSA, enterococci, Pseudomonas aeruginosa) is declining, as it is among pneumococci and enteropathogens; but it is rising among community-acquired, uropathogenic Escherichia coli. Resistance to colistin is slowly increasing; and worrisome resistance determinants, such as blaNDM-1 and mcr-1, appeared in Mexico shortly after their first report elsewhere. After four years from the United Nations General Assembly high-level meeting on antibiotic resistance, Mexico is yet to deploy the basic measures to assess and control antibiotic resistance. As such, and despite the regulatory changes, the 2010-2020 period looks like a "lost decade".

Selection of Amikacin Hyper-Resistant Pseudomonas aeruginosa After Stepwise Exposure to High Amikacin Concentrations.

Aerosolized amikacin reaches high concentrations in lung fluids, which are well above the minimum inhibitory concentrations (MICs) of resistant strains of Pseudomonas aeruginosa. However, P. aeruginosa can gain resistance to amikacin through different cumulative mechanisms; amikacin MICs are seldom reported beyond values of 1,000 µg/ml, as tested in clinical microbiology assays. To assess how high amikacin MICs can be reached by graded exposure, four amikacin-resistant P. aeruginosa isolates were grown in a 4-step increased exposure to amikacin; derivative strains were further characterized by measuring their comparative growth rate, biofilm-forming ability, and susceptibility to other antibiotics. In addition, the mechanism underlying the MIC increase was assessed phenotypically, using a set of 12 aminoglycoside disks, and measuring the effect of Phe-Arg-ß-naphthylamide, an efflux pump inhibitor. Graded exposure to amikacin increased MICs of resistant strains up to 10,000-20,000 µg/ml, without apparent fitness cost, and having variable consequences on their biofilm-forming ability, and on their susceptibility to other antibiotics. Decreased permeability may have contributed to hyper-resistance, although evidence was inconclusive and variable between strains. Amikacin-resistant P. aeruginosa is able to gain in vitro hyper-resistance with minimal changes in the specific phenotypes that were tested; the ability to achieve high-level amikacin (AMK) resistance may confound the clinical utility of this aerosolized AMK, but clinical data would be required to assess this.

Moxifloxacin versus levofloxacin against acute exacerbations of chronic bronchitis: the Latin American Cohort.

We compared the efficacy and safety of moxifloxacin and levofloxacin for the treatment of patients with acute exacerbations of chronic bronchitis (AECB) using a prospective, randomized, double blind, parallel-group clinical trial design. A total of 563 patients with AECB were enrolled (437 efficacy-valid) at 34 centers in Mexico, Argentina, Brazil, Colombia, and Peru. Patients were randomized to oral therapy with either moxifloxacin 400 mg once daily for 5 days or levofloxacin 500 mg once daily for 7 days. Clinical success was achieved in 201 out of 221 (91.0%) patients in the moxifloxacin group, and in 203 out of 216 (94.0%) in the levofloxacin group, indicating that moxifloxacin is equivalently effective to levofloxacin. Bacteriologic eradication or presumed eradication was also similar in the two treatment groups: 92.8% in the moxifloxacin group and 93.8% in the levofloxacin group. Nausea was the most common drug-related adverse event in each treatment group. The rate of discontinuation because of adverse events was very low (2%). In conclusion, a 5-day course of moxifloxacin is clinically and bacteriologically equivalent to a 7-day course of levofloxacin in the treatment of patients with AECB. The short treatment duration with moxifloxacin may have compliance advantages over other currently used therapies in the 'real-life' clinical setting.

Urban dust fecal pollution in Mexico City: antibiotic resistance and virulence factors of Escherichia coli.

Fecal pollution of settled dust samples from indoor and outdoor urban environments, was measured and characterized by the presence of fecal coliforms (FC), and by the characterization of Escherichia coli virulence genes, adherence and antibiotic resistance traits as markers. There were more FC indoors than outdoors (mean values 1089 and 435MPN/g). Among indoor samples, there were more FC in houses with carpets and/or pets. Using a PCR-based assay for six enteropathogenicity genes (belonging to the EAEC, EHEC and EPEC pathotypes) on randomly selected E. coli isolates, there was no significant difference between isolates from indoors and outdoors (60% and 72% positive to at least one gene). The serotypes commonly associated with pathogenic strains, such as O86 and O28, were found in the indoor isolates; whereas O4, O66 and O9 were found amongst outdoor isolates. However, there were significantly more outdoor isolates resistant to at least one antibiotic (73% vs. 45% from indoors) among the strains positive for virulence factors, and outdoor isolates were more commonly multiresistant. There was no relationship between the presence of virulence genes and resistance traits. These results indicate that outdoor fecal bacteria were more likely from human sources, and those found indoors were related to pets and maintained in carpets. This study illustrates the risk posed by fecal bacteria from human sources, usually bearing virulence and resistance traits. Furthermore, the high prevalence of strains carrying genes associated to EAEC or EHEC pathotypes, in both indoor and outdoor environments, adds to the health risk.

Sublethal exposure to commercial formulations of the herbicides dicamba, 2,4-dichlorophenoxyacetic acid, and glyphosate cause changes in antibiotic susceptibility in Escherichia coli and Salmonella enterica serovar Typhimurium.

UNLABELLED: Biocides, such as herbicides, are routinely tested for toxicity but not for sublethal effects on microbes. Many biocides are known to induce an adaptive multiple-antibiotic resistance phenotype. This can be due to either an increase in the expression of efflux pumps, a reduced synthesis of outer membrane porins, or both. Exposures of Escherichia coli and Salmonella enterica serovar Typhimurium to commercial formulations of three herbicides-dicamba (Kamba), 2,4-dichlorophenoxyacetic acid (2,4-D), and glyphosate (Roundup)-were found to induce a changed response to antibiotics. Killing curves in the presence and absence of sublethal herbicide concentrations showed that the directions and the magnitudes of responses varied by herbicide, antibiotic, and species. When induced, MICs of antibiotics of five different classes changed up to 6-fold. In some cases the MIC increased, and in others it decreased. Herbicide concentrations needed to invoke the maximal response were above current food maximum residue levels but within application levels for all herbicides. Compounds that could cause induction had additive effects in combination. The role of soxS, an inducer of the AcrAB efflux pump, was tested in ß-galactosidase assays with soxS-lacZ fusion strains of E. coli. Dicamba was a moderate inducer of the sox regulon. Growth assays with Phe-Arg ß-naphtylamide (PAßN), an efflux pump inhibitor, confirmed a significant role of efflux in the increased tolerance of E. coli to chloramphenicol in the presence of dicamba and to kanamycin in the presence of glyphosate. Pathways of exposure with relevance to the health of humans, domestic animals, and critical insects are discussed. IMPORTANCE: Increasingly common chemicals used in agriculture, domestic gardens, and public places can induce a multiple-antibiotic resistance phenotype in potential pathogens. The effect occurs upon simultaneous exposure to antibiotics and is faster than the lethal effect of antibiotics. The magnitude of the induced response may undermine antibiotic therapy and substantially increase the probability of spontaneous mutation to higher levels of resistance. The combination of high use of both herbicides and antibiotics in proximity to farm animals and important insects, such as honeybees, might also compromise their therapeutic effects and drive greater use of antibiotics. To address the crisis of antibiotic resistance requires broadening our view of environmental contributors to the evolution of resistance.

Antibiotic resistance in oral commensal streptococci from healthy Mexicans and Cubans: resistance prevalence does not mirror antibiotic usage.

Antibiotic resistance genes might be maintained by selection pressures different from those which are responsible for initially selecting resistant bacteria. This possibility was suggested from a comparison of oral commensal streptococci isolated from healthy people not taking antibiotics. Resistance frequencies were similar for organisms from Mexico and Cuba despite significant differences in antibiotic usage in these two countries. Resistance to > or = 4 drugs was far more common in Mexico, the only detectable trend that can be related to the higher use of antibiotics in Mexico. If resistance is not uniquely maintained by antibiotics, then other environmental factors must also be at work. These need to be identified if a strategy to control antibiotic resistance is to be successful.

Comparison of antibiotic susceptibility and plasmid content, between biofilm producing and non-producing clinical isolates of Pseudomonas aeruginosa.

Bacteria growing as biofilms are less susceptible to antimicrobial agents than free-living cells. Several infectious processes are now recognised to be related to biofilm formation. Pseudomonas aeruginosa is a known biofilm-producing organism and often resistant to several antibiotics. To assess the relationship between biofilm-forming capabilities and antibiotic resistance phenotypes in P. aeruginosa clinical isolates, we evaluated the ability of different phenotypes to form biofilms using 162 clinical isolates of P. aeruginosa. Only 14% of strains produced biofilm after an 8-h incubation and 8% after 24h; the latter group was slightly more multi-resistant, particularly to piperacillin-tazobactam and imipenem. The frequency of 8-h biofilm-formers was higher among isolates resistant to four agents, and that of 24-h biofilm-formers higher among isolates resistant to six antibiotics. Plasmid-content was not related to biofilm formation. In addition to the protection against antibiotics that bacteria gain by growing in a biofilm, clinical isolates forming biofilms seem to accumulate more resistance phenotypes. Bacteria in biofilms may survive antibiotic exposure not only because of the protective effect of biofilm itself, but also because of the increased frequency of resistance traits present.

El efecto inhibitorio del EDTA sobre el crecimiento microbiano en suspensiones de propofol / The inhibitory effect of EDTA on microbial growth in propofol suspensions

Resumen: Las suspensiones comerciales de propofol, por su composición farmacéutica, soportan el crecimiento de diversos microorganismos; la aplicación de propofol, contaminado microbianamente luego de ser retirado de su envase original, ha sido vinculada a brotes de infección postoperatoria. La adición de sales de ácido etilendiaminotetraacético (EDTA) retarda el crecimiento de estos microorganismos. Aquí se comparó el crecimiento, a lo largo de 48 horas y en tres temperaturas (ambiente, 35 y 42 oC), de siete cepas bacterianas y tres de levaduras, en cuatro formulaciones de propofol disponibles en México, una de ellas adicionada con EDTA. Consistentemente, el crecimiento fue menor en la suspensión con EDTA, comparada con las tres que no lo contienen, con variaciones entre microorganismos y temperaturas: desde muerte inicial de parte del inóculo, o inhibición completa y sostenida del crecimiento, hasta inhibición parcial. Aunque la adición de EDTA no debe considerarse como un sustituto del manejo aséptico del propofol, que debe extenderse durante el período perioperatorio, ciertamente disminuye la proliferación microbiana que puede darse por contaminación accidental, disminuyendo asimismo el riesgo de infección para el paciente.

Abstract: Commercially available propofol suspensions, due to their pharmaceutical composition, support the growth of several microorganisms; the administration of propofol suspensions that became microbially-contaminated after being removed from their original vial, has been linked to postsurgical infections. Addition of ethylenediaminetetracetic acid (EDTA) salts delays the growth of such microorganisms. Here, we compared the growth of seven bacterial strains and three yeast strains, along 48 hours and at three different incubation temperatures (room temperature, 35 and 42 oC), in four propofol formulations available in Mexico, one of them with supplemented EDTA. Consistently, microbial growth was diminished in the formulation supplemented with EDTA, compared to the other three, although with variations between microorganisms and incubation temperatures: from initial reduction in viable organisms, to complete and sustained growth inhibition, to only partial growth inhibition. While the addition of EDTA to propofol suspensions must not be considered as a substitute for aseptic handling of the drug, it certainly diminishes microbial growth that can occur after accidental contamination, reducing the infection risk for the patient.

Resistance to antibiotics of clinical relevance in the fecal microbiota of Mexican wildlife.

There are a growing number of reports of antibiotic resistance (ATBR) in bacteria living in wildlife. This is a cause for concern as ATBR in wildlife represents a potential public health threat. However, little is known about the factors that might determine the presence, abundance and dispersion of ATBR bacteria in wildlife. Here, we used culture and molecular methods to assess ATBR in bacteria in fecal samples from howler monkeys (Alouatta palliata), spider monkeys (Ateles geoffroyi), tapirs (Tapirus bairdii) and felids (jaguars, Panthera onca; pumas, Puma concolor; jaguarundis, Puma yagouaroundi; and ocelots, Leopardus pardalis) living freely in two regions of the Mexican state of Veracruz under different degrees of human influence. Overall, our study shows that ATBR is commonplace in bacteria isolated from wildlife in southeast Mexico. Most of the resistances were towards old and naturally occurring antibiotics, but we also observed resistances of potential clinical significance. We found that proximity to humans positively affected the presence of ATBR and that ATBR was higher in terrestrial than arboreal species. We also found evidence suggesting different terrestrial and aerial routes for the transmission of ATBR between humans and wildlife. The prevalence and potential ATBR transfer mechanisms between humans and wildlife observed in this study highlight the need for further studies to identify the factors that might determine ATBR presence, abundance and distribution.