Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.     

Antibiotic sensitivities of Neisseria gonorrhoeae in the Toronto area

The antibiotic sensitivity pattern of 3872 isolates of N. gonorrhoeae tested in Toronto from 1969 to 1973 is reviewed. An increase in resistance to both penicillin and tetracycline was noted up to 1971, but no further increase has occurred since then. Ninety-seven percent of 135 patients with “sensitive” strains (inhibited by 0.3 U/ml of penicillin and/or 0.5 μg/ml of tetracycline) were cured by either 8 g of tetracycline or 5,000,000 U of penicillin, whereas only 59% of 58 patients with “resistant” strains (requiring 1.0 U/ml of penicillin and/or 2.0 μg/ml of tetracycline for inhibition) were cured by the same dosage. Spectinomycin appears to be an acceptable alternative therapy. Maximum doses of the chosen drug are recommended in the hope of retarding further spread of more resistant organisms.     

The Challenge of Antibiotic-Resistant Staphylococcus: Lessons from Hospital Nurseries in the mid-20th Century

In the late 1940s, epidemics of antibiotic-resistant strains of Staphylococcus aureus began to plague postpartum nurseries in hospitals across the United States. Exacerbated by overcrowding and nursing shortages, resistant S. aureus outbreaks posed a novel challenge to physicians and nurses heavily reliant on antibiotics as both prophylaxis and treatment. This paper explores the investigation of the reservoir, mode of transmission, and virulence of S. aureus during major hospital outbreaks and the subsequent implementation of novel infection control measures from the late 1940s through the early 1960s. The exploration of these measures reveals a shift in infection control policy as hospitals, faced with the failure of antibiotics to slow S. aureus outbreaks, implemented laboratory culture routines, modified nursery structure and layout, and altered nursing staff procedures to counter various forms of S. aureus transmission. Showcasing the need for widespread epidemiologic surveillance, ultimately manifesting itself in specialized “hospital epidemiology” training promoted in the 1970s, the challenges faced by hospital nurses in the 1950s prove highly relevant to the continued struggle with methicillin-resistant Staphylococcus aureus (MRSA) and other resistant nosocomial infections.     

Chicken Cathelicidins Display Antimicrobial Activity against Multiresistant Bacteria without Inducing Strong Resistance

The increased prevalence of multidrug-resistant (MDR) bacteria in combination with the relatively limited development of new antibiotics presents a serious threat to public health. In chicken, especially Extended-Spectrum ß-Lactamase (ESBL) carrying Enterobacteriaceae are often asymptomatically present but can infect humans. Due to their broad range antimicrobial activity cathelicidins and other host defence peptides, are considered to be an attractive alternative to conventional antibiotics. In this study, the antimicrobial activity of three chicken cathelicidins against a broad array of multidrug resistant bacteria was determined. All three peptides showed high antibacterial activity independent of the presence of MDR characteristics. Induction experiments using S. aureus and K. pneumoniae showed that although an increase in resistance was initially observed, susceptibility towards chicken cathelicidins remained high and no major resistance was developed. The combined results underline the potential of chicken cathelicidins as a new alternative to antibiotics.     

High antimicrobial resistance among bacterial isolates of blood stream infections (BSI) in a Nigerian University Teaching Hospital

The antimicrobial resistance profile of 220 bacteria isolated from 1,006 episodes of blood stream infections (BSI) between January 2004 and December 2005 in a University Teaching Hospital, Southwestern Nigeria, were analyzed. Gram positive bacteria constituted 47.3% while Gram negative constituted 52.7%. The most common organisms were Staphylococcus aureus (37.3%), Klebsiella (30%), Pseudomonas (8.2%), Proteus (6.4%), Escherichia coli (5.5%) and coagulase negative staphylococci (4.6%). The cumulative resistance of all the bacteria isolates to ampicillin was 79%, gentamicin 51%, ceftazidime 11% and ciprofloxacin 6%. About 85% of the Gram positive bacteria were resistant to penicillinG, 79% to methicillin and 37% to erythromycin while 74% of the Gram negative bacteria were resistant to cotrimoxazole, 69% to tetracycline and 38% to chloramphenicol. Among the 7 antibiotics tested for each group, 7 patterns of antibiotic resistance were observed for each; 6 were multi-drug pattern with number of antibiotics ranging from 2 to 7. This study demonstrates high antimicrobial resistance among clinical bacterial isolates of BSI to commonly prescribed antibiotics most especially penicillinG, ampicillin, methicillin, cotrimoxazole, tetracycline and gentamicin. Based on the result of this study, it is suggested that the combination of ampicillin and gentamicin normally employed for empirical treatment of BSI in our hospital should be stopped.     

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.     

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla _NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla _NDM-1 Klebsiella pneumonia and bla _NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla _NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.     

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.     

Alteration of 23 S ribosomal RNA and erythromycin-induced resistance to lincomycin and spiramycin in Staphylococcus aureus

Functionally active “hybrid” 50 S ribosomal subunits can be reconstituted using 23 S RNA from Staphylococcus aureus (strain 1206) and 5 S RNA, as well as 50 S ribosomal proteins from Bacillus stearothermophilus. Using this system, resistance of S. aureus 50 S subunits to lincomycin and spiramycin was analyzed. When 23 S RNA from either phenotypically resistant (“induced resistance”) S. aureuscells or derived genetically resistant (“constitutive resistance”) S. aureus cells, were used, the reconstituted 50 S subunits showed the resistant phenotype similar to that seen in native 50 S subunits obtained from resistant cells; only very weak inhibition by the antibiotics was observed in poly (U) - directed polyphenylalanine synthesis involving these 50 S subunits. In contrast, the 50 S particles reconstituted using 23 S RNA from uninduced (sensitive) S. aureus were subject to greater inhibition by the antibiotics in cell-free poly-peptide synthesis. It is concluded that modification of 23 S RNA, presumably the previously observed methylation to form dimethyladenine, is responsible for the resistance to the antibiotics in this strain of S. aureus.     

Sporicidal properties of hydrogen peroxide against food spoilage organisms

The sporicidal properties of hydrogen peroxide were evaluated at concentrations of 10 to 41% and at temperatures of 24 to 76 C. The organisms tested and their relative resistance at 24 C to 25.8% H₂O₂ were: Bacillus subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus > Clostridium sp. putrefactive anaerobe 3679 > S. aureus, with „D” values of 7.3, 2, 1.8, 1.5, 0.8., and 0.2 min, respectively. Heat shocking spores prior to hydrogen peroxide treatment decreased their resistance. Wet spores were more resistant than dry spores when good mixing was achieved during hydrogen peroxide treatment. Inactivation curves followed first-order kinetics except for a lag period where the inactivation rate was very slow. Increasing the H₂O₂ concentration and the temperature reduced the lag period.     

Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina

Background

There has been considerable effort to discover plant-derived antibacterials against methicillin-resistant strains of Staphylococcus aureus (MRSA) which have developed resistance to most existing antibiotics, including the last line of defence, vancomycin. Pentacyclic triterpenoid, a biologically diverse plant-derived natural product, has been reported to show anti-staphylococcal activities. The objective of this study is to evaluate the interaction between three pentacyclic triterpenoid and standard antibiotics (methicillin and vancomycin) against reference strains of Staphylococcus aureus.

Methods and Results

The activity of the standard antibiotics and compounds on reference methicillin-sensitive and resistant strains of S. aureus were determined using the macrodilution broth method. The minimum inhibitory concentration (MIC) of the compounds was compared with that of the standard antibiotics. The interaction between any two antimicrobial agents was estimated by calculating the fractional inhibitory concentration (FIC index) of the combination. The various combinations of antibiotics and compounds reduced the MIC to a range of 0.05 to 50%.

Conclusion

Pentacyclic triterpenoids have shown anti-staphylococcal activities and although individually weaker than common antibiotics produced from bacteria and fungi, synergistically these compounds may use different mechanism of action or pathways to exert their antimicrobial effects, as implicated in the lowered MICs. Therefore, the use of current antibiotics could be maintained in their combination with plant-derived antibacterial agents as a therapeutic option in the treatment of S. aureus infections.     

Sublethal vancomycin-induced ROS mediating antibiotic resistance in Staphylococcus aureus

Staphylococcus aureus is the leading cause of many human infectious diseases. Besides infectious dangers, S. aureus is well-known for the quickly developed drug resistance. Although great efforts have been made, mechanisms underlying the antibiotic effects of S. aureus are still not well clarified. Recently, reports have shown that oxidative stress connects with bactericidal antibiotics [Dwyer et al. (2009) Curr. Opin. Microbiol. 12, 482–489]. Based on this point, we demonstrate that reactive oxygen species (ROS) induced by sublethal vancomycin may be partly responsible for the antibiotic resistance in heterogeneous vancomycin resistant S. aureus (hVRSA). Sublethal vancomycin treatment may induce protective ROS productions in hVRSA, whereas reduction in ROS level in hVRSA strains may increase their vancomycin susceptibility. Moreover, low dose of ROS in VSSA (vancomycin susceptible S. aureus) strains may promote their survival under vancomycin conditions. Our findings reveal that modest ROS generation may be protective for vancomycin resistance in hVRSA. These results recover novel insights into the relationship between oxidative stress and bacterial resistance, which has important applications for further use of antibiotics and development of therapeutics strategies for hVRSA.     

No Outbreak of Vancomycin and Linezolid Resistance in Staphylococcal Pneumonia over a 10-Year Period

Background

Staphylococci can cause wound infections and community- and nosocomial-acquired pneumonia, among a range of illnesses. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) have been rapidly increasing as a cause of infections worldwide in recent decades. Numerous reports indicate that S. aureus and MRSA are becoming resistant to many antibiotics, which makes them very dangerous. Therefore, this study retrospectively investigated the resistance to antimicrobial agents in all hospitalized patients suffering from community- or nosocomial-acquired pneumonia due to S. aureus and MRSA.

Methods

Information from the study groups suffering from either community- or nosocomial-acquired pneumonia caused by S. aureus or MRSA was gathered by searching records from 2004 to 2014 at the HELIOS Clinic Wuppertal, Witten/Herdecke University, Germany. The findings of antibiotic resistance were analyzed after the evaluation of susceptibility testing for S. aureus and MRSA.

Results

Total of 147 patients (63.9%, 95% CI 57.5%–69.8%), mean age 67.9 ± 18.5 years, with pneumonia triggered by S. aureus, and 83 patients (36.1%, 95% CI 30.2%–42.5%), mean age 72.3 ± 13.8 years, with pneumonia due to MRSA. S. aureus and MRSA developed no resistance to vancomycin (P = 0.019 vs. < 0.0001, respectively) or linezolid (P = 0.342 vs. < 0.0001, respectively). MRSA (95.3%) and S. aureus (56.3%) showed a high resistance to penicillin. MRSA (87.7%) was also found to have a high antibiotic resistance against ß-lactam antibiotics, compared to S. aureus (9.6%). Furthermore, MRSA compared to S. aureus, respectively, had increased antibiotic resistance to ciprofloxacin (90.1% vs. 17.0%), cefazolin (89.7% vs. 10.2%), cefuroxime (89.0% vs. 9.1%), levofloxacin (88.2% vs. 18.4%), clindamycin (78.0% vs. 14.7%), and erythromycin (76.5% vs. 20.8%).

Conclusion

No development of resistance was found to vancomycin and linezolid in patients with pneumonia caused by S. aureus and MRSA.     

Transferable antibiotic resistance in E. coli and Klebsiella pneumoniae

Twenty-three of 43 E. coli and 25 of 39 Klebsiella isolates, resistant to two or more antibiotics, transferred one or more resistance genes to a recipient E. coli K₁₂ culture. Resistances transferred most frequently by both species were those to kanamycin and neomycin. E. coli cultures transferred resistance to tetracycline, chloramphenicol, ampicillin and carbenicillin, whereas Klebsiella isolates transferred resistance to the first two of these antibiotics. Extrapolation of these results to a larger series of isolations of E. coli and Klebsiella from hospital patients suggested that 21 and 18% respectively of cultures of these two organisms carried potentially transferable resistance.     

Novel Method for Rapid Assessment of Antibiotic Resistance in Escherichia coli Isolates from Environmental Waters by Use of a Modified Chromogenic Agar

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ± 3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.     

Insecticide Mixtures Could Enhance the Toxicity of Insecticides in a Resistant Dairy Population of Musca domestica L

House flies, Musca domestica L., are important pests of dairy operations worldwide, with the ability to adapt wide range of environmental conditions. There are a number of insecticides used for their management, but development of resistance is a serious problem. Insecticide mixtures could enhance the toxicity of insecticides in resistant insect pests, thus resulting as a potential resistance management tool. The toxicity of bifenthrin, cypermethrin, deltamethrin, chlorpyrifos, profenofos, emamectin benzoate and fipronil were assessed separately, and in mixtures against house flies. A field-collected population was significantly resistant to all the insecticides under investigation when compared with a laboratory susceptible strain. Most of the insecticide mixtures like one pyrethroid with other compounds evaluated under two conditions (1∶1-“A” and LC₅₀: LC₅₀-“B”) significantly increased the toxicity of pyrethroids in the field population. Under both conditions, the combination indices of pyrethroids with other compounds, in most of the cases, were significantly below 1, suggesting synergism. The enzyme inhibitors, PBO and DEF, when used in combination with insecticides against the resistant population, toxicities of bifenthrin, cypermethrin, deltamethrin and emamectin were significantly increased, suggesting esterase and monooxygenase based resistance mechanism. The toxicities of bifenthrin, cypermethrin and deltamethrin in the resistant population of house flies could be enhanced by the combination with chlorpyrifos, profenofos, emamectin and fipronil. The findings of the present study might have practical significance for resistance management in house flies.     

Hp1404, a New Antimicrobial Peptide from the Scorpion Heterometrus petersii

Antimicrobial peptides have attracted much interest as a novel class of antibiotics against a variety of microbes including antibiotics resistant strains. In this study, a new cationic antimicrobial peptide Hp1404 was identified from the scorpion Heterometrus petersii, which is an amphipathic α-helical peptide and has a specific inhibitory activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. Hp1404 can penetrate the membrane of S. aureus at low concentration, and disrupts the cellular membrane directly at super high concentration. S. aureus does not develop drug resistance after multiple treatments with Hp1404 at sub MIC concentration, which is possibly associated with the antibacterial mechanism of the peptide. In addition, Hp1404 has low toxicity to both mammalian cells (HC₅₀  =  226.6 µg/mL and CC₅₀ > 100 µg/mL) and balb-c mice (Non-toxicity at 80 mg/Kg by intraperitoneal injection and LD₅₀  =  89.8 mg/Kg by intravenous injection). Interestingly, Hp1404 can improve the survival rate of the MRSA infected balb-c mice in the peritonitis model. Taken together, Hp1404 may have potential applications as an antibacterial agent.     

The Mechanism of Heterogeneous Beta-Lactam Resistance in MRSA: Key Role of the Stringent Stress Response

All methicillin resistant S. aureus (MRSA) strains carry an acquired genetic determinant – mecA or mecC - which encode for a low affinity penicillin binding protein –PBP2A or PBP2A′ – that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique – heterogeneous – phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA) that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response.     

Antimicrobials, drug discovery, and genome mining

Over the years, antibiotics have provided an effective treatment for a number of microbial diseases. However recently, there has been an increase in resistant microorganisms that have adapted to our current antibiotics. One of the most dangerous pathogens is methicillin-resistant Staphylococcus aureus (MRSA). With the rise in the cases of MRSA and other resistant pathogens such as vancomycin-resistant Staphylococcus aureus, the need for new antibiotics increases every day. Many challenges face the discovery and development of new antibiotics, making it difficult for these new drugs to reach the market, especially since many of the pharmaceutical companies have stopped searching for antibiotics. With the advent of genome sequencing, new antibiotics are being found by the techniques of genome mining, offering hope for the future.