Antimicrobial Resistance in Equine Reproduction

Bacteria develop resistance to antibiotics following low-level “background” exposure to antimicrobial agents as well as from exposure at therapeutic levels during treatment for bacterial infections. In this review, we look specifically at antimicrobial resistance (AMR) in the equine reproductive tract and its possible origin, focusing particularly on antibiotics in semen extenders used in preparing semen doses for artificial insemination. Our review of the literature indicated that AMR in the equine uterus and vagina were reported worldwide in the last 20 years, in locations as diverse as Europe, India, and the United States. Bacteria colonizing the mucosa of the reproductive tract are transferred to semen during collection; further contamination of the semen may occur during processing, despite strict attention to hygiene at critical control points. These bacteria compete with spermatozoa for nutrients in the semen extender, producing metabolic byproducts and toxins that have a detrimental effect on sperm quality. Potential pathogens such as Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa may occasionally cause fertility issues in inseminated mares. Antibiotics are added during semen processing, according to legislation, to impede the growth of these microorganisms but may have a detrimental effect on sperm quality, depending on the antimicrobial agent and concentration used. However, this addition of antibiotics is counter to current recommendations on the prudent use of antibiotics, which recommend that antibiotics should be used only for therapeutic purposes and after establishing bacterial sensitivity. There is some evidence of resistance among bacteria found in semen samples. Potential alternatives to the addition of antibiotics are considered, especially physical removal separation of spermatozoa from bacteria. Suggestions for further research with colloid centrifugation are provided.

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Need for Annual Surveillance of Antimicrobial Resistance in Streptococcus pneumoniae in the United States: 2-Year Longitudinal Analysis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1037-1042.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1037-1042 ◽

Cited By ~ 94

Author(s):

Daniel F. Sahm ◽

James A. Karlowsky ◽

Laurie J. Kelly ◽

Ian A. Critchley ◽

Mark E. Jones ◽

...

Keyword(s):

United States ◽

Streptococcus Pneumoniae ◽

Antimicrobial Resistance ◽

Antimicrobial Agents ◽

The United States ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Resistance Patterns ◽

Changing Patterns ◽

Resistance To Penicillin

ABSTRACT Although changing patterns in antimicrobial resistance inStreptococcus pneumoniae have prompted several surveillance initiatives in recent years, the frequency with which these studies are needed has not been addressed. To approach this issue, the extent to which resistance patterns change over a 1-year period was examined. In this study we analyzed S. pneumoniaeantimicrobial susceptibility results produced in our laboratory with isolates obtained over 2 consecutive years (1997–1998 and 1998–1999) from the same 96 institutions distributed throughout the United States. Comparison of results revealed increases in resistant percentages for all antimicrobial agents studied except vancomycin. For four of the agents tested (penicillin, cefuroxime, trimethoprim-sulfamethoxazole, and levofloxacin), the increases were statistically significant (P < 0.05). Resistance to the fluoroquinolone remained low in both years (0.1 and 0.6%, respectively); in contrast, resistance to macrolides was consistently greater than 20%, and resistance to trimethoprim-sulfamethoxazole increased from 13.3 to 27.3%. Multidrug resistance, concurrent resistance to three or more antimicrobials of different chemical classes, also increased significantly between years, from 5.9 to 11%. The most prevalent phenotype was resistance to penicillin, azithromycin (representative macrolide), and trimethoprim-sulfamethoxazole. Multidrug-resistant phenotypes that included fluoroquinolone resistance were uncommon; however, two phenotypes that included fluoroquinolone resistance not found in 1997–1998 were encountered in 1998–1999. This longitudinal surveillance study of resistance inS. pneumoniae revealed that significant changes do occur in just a single year and supports the need for surveillance at least on an annual basis, if not continuously.

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Crumbling the Castle: Targeting DNABII Proteins for Collapsing Bacterial Biofilms as a Therapeutic Approach to Treat Disease and Combat Antimicrobial Resistance

Antibiotics ◽

10.3390/antibiotics11010104 ◽

2022 ◽

Vol 11 (1) ◽

pp. 104

Author(s):

James V. Rogers ◽

Veronica L. Hall ◽

Charles C. McOsker

Keyword(s):

Antimicrobial Resistance ◽

Bacterial Infections ◽

Antimicrobial Agents ◽

Defense Mechanism ◽

Treatment Options ◽

Bacterial Biofilms ◽

Host Immune System ◽

New Antibiotics ◽

Financial Burdens ◽

Global Threat

Antimicrobial resistance (AMR) is a concerning global threat that, if not addressed, could lead to increases in morbidity and mortality, coupled with societal and financial burdens. The emergence of AMR bacteria can be attributed, in part, to the decreased development of new antibiotics, increased misuse and overuse of existing antibiotics, and inadequate treatment options for biofilms formed during bacterial infections. Biofilms are complex microbiomes enshrouded in a self-produced extracellular polymeric substance (EPS) that is a primary defense mechanism of the resident microorganisms against antimicrobial agents and the host immune system. In addition to the physical protective EPS barrier, biofilm-resident bacteria exhibit tolerance mechanisms enabling persistence and the establishment of recurrent infections. As current antibiotics and therapeutics are becoming less effective in combating AMR, new innovative technologies are needed to address the growing AMR threat. This perspective article highlights such a product, CMTX-101, a humanized monoclonal antibody that targets a universal component of bacterial biofilms, leading to pathogen-agnostic rapid biofilm collapse and engaging three modes of action—the sensitization of bacteria to antibiotics, host immune enablement, and the suppression of site-specific tissue inflammation. CMTX-101 is a new tool used to enhance the effectiveness of existing, relatively inexpensive first-line antibiotics to fight infections while promoting antimicrobial stewardship.

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Antimicrobial Resistance in Salmonella in the United States from 1948 to 1995

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02536-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2567-2571 ◽

Cited By ~ 17

Author(s):

Daniel A. Tadesse ◽

Aparna Singh ◽

Shaohua Zhao ◽

Mary Bartholomew ◽

Niketta Womack ◽

...

Keyword(s):

United States ◽

Retrospective Study ◽

Multidrug Resistance ◽

Antimicrobial Resistance ◽

Salmonella Enterica ◽

Antimicrobial Agents ◽

The United States ◽

Content Type ◽

The Past ◽

The 1950S

ABSTRACTWe conducted a retrospective study of 2,149 clinicalSalmonellastrains to help document the historical emergence of antimicrobial resistance. There were significant increases in resistance to older drugs, including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, which were most common inSalmonella entericaserotype Typhimurium. An increase in multidrug resistance was observed for each decade since the 1950s. These data help show howSalmonellaevolved over the past 6 decades, after the introduction of new antimicrobial agents.

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Innovative approaches to treat Staphylococcus aureus biofilm-related infections

Essays in Biochemistry ◽

10.1042/ebc20160056 ◽

2017 ◽

Vol 61 (1) ◽

pp. 61-70 ◽

Cited By ~ 17

Author(s):

Katharina Richter ◽

Freija Van den Driessche ◽

Tom Coenye

Keyword(s):

Staphylococcus Aureus ◽

Extracellular Matrix ◽

Antimicrobial Resistance ◽

Iron Metabolism ◽

Communication System ◽

Bacterial Infections ◽

Antimicrobial Agents ◽

Antibiofilm Activity ◽

Human Pathogen ◽

Bacterial Communication

Many bacterial infections in humans and animals are caused by bacteria residing in biofilms, complex communities of attached organisms embedded in an extracellular matrix. One of the key properties of microorganisms residing in a biofilm is decreased susceptibility towards antimicrobial agents. This decreased susceptibility, together with conventional mechanisms leading to antimicrobial resistance, makes biofilm-related infections increasingly difficult to treat and alternative antibiofilm strategies are urgently required. In this review, we present three such strategies to combat biofilm-related infections with the important human pathogen Staphylococcus aureus: (i) targeting the bacterial communication system with quorum sensing (QS) inhibitors, (ii) a ‘Trojan Horse’ strategy to disturb iron metabolism by using gallium-based therapeutics and (iii) the use of ‘non-antibiotics’ with antibiofilm activity identified through screening of repurposing libraries.

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Critically important antimicrobial preparations for veterinary medicine

Scientific Messenger of LNU of Veterinary Medicine and Biotechnology ◽

10.15421/nvlvet8704 ◽

2018 ◽

Vol 20 (87) ◽

pp. 19-26 ◽

Cited By ~ 1

Author(s):

T.I. Stetsko ◽

V.P. Muzyka ◽

V.M. Hunchak

Keyword(s):

Veterinary Medicine ◽

Antimicrobial Resistance ◽

Antimicrobial Therapy ◽

Bacterial Infections ◽

Antimicrobial Agents ◽

Animal Health ◽

Point Of View ◽

Animal Origin ◽

European Medicines Agency ◽

Antimicrobial Substances

The resistance of microorganisms, bacterial pathogens, to antimicrobials is a global problem in both healthcare and veterinary medicine. It is believed that the main reason for the emergence and spread of antimicrobial resistance in humans is the transfer of antibiotic resistant strains of microorganisms or genes, determinants of resistance, through products of animal origin from productive animals to humans. Thus, the main way of antimicrobial resistance containment is to restrain and minimize it through the prudent use of antibiotics in veterinary medicine, especially those that are critically important for productive animals. In addition, some classes of antibacterial substances and antibiotics, that are widely used in humane medicine, are used in veterinary medicine. The need to use and preserve these important therapeutic agents is relevant from the point of view of the concept «One Health». The article provides a list of all antibacterial substances used by productive animals for their importance in veterinary medicine, developed by a special group of experts of the World Organisation for Animal Health (OIE). Any antimicrobial agent authorized for use in veterinary medicine for productive animals, in accordance with the criteria for quality, safety and efficacy as defined in Section 6.9 of the Terrestrial Animal Health Code, is considered to be important for veterinary medicine. All the antimicrobial substances used for productive animals are divided in this list on critical, very important and important for veterinary medicine. Attention was also drawn to the peculiarities of the use of critical antimicrobial agents in veterinary medicine, especially those recognized as critical in humane medicine. These include aminoglycosides, cephalosporins of the 3rd and 4th generation, fluoroquinolones, glycopeptides, macrolides, some penicillins and polymyxins. The article also describes the classification of critical antimicrobials by the European Medicines Agency (EMA) and the Panel of Experts on Antimicrobials (AMEG) of the WHO based on the risk profile for humans through the development of antimicrobial resistance after application to productive animals. Such an assessment will give veterinary practitioners an important justification when they make decisions about the clinical treatment of bacterial infections and the responsible appointment of antimicrobial therapy. This will help to reach the balance among the achievement of the effectiveness of antimicrobial therapy of productive animals, reducing of the selective pressure on the development of antibiotic resistance and ensuring of a high level of human health.

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MULTIDRUG RESISTANCE (MDR) OF V. Parahaemolyticus

Jurnal Riset Kimia ◽

10.25077/jrk.v2i2.150 ◽

2015 ◽

Vol 2 (2) ◽

pp. 112

Author(s):

Marlina

Keyword(s):

United States ◽

Antimicrobial Resistance ◽

Vibrio Parahaemolyticus ◽

Antimicrobial Agents ◽

The United States ◽

Food Microbiology ◽

Aeromonas Species ◽

General Strategy ◽

Rapd Pcr

Vol. 2, No. 2 ABSTRACT A total of 97 V. parahaemolyticus isolate from Padang were examined for their resistance to 15 antibiotics. V. parahaemolyticus isolated behaved as resistant to sulfamethoxazole (100%), rifampin (95%) and tetracycline (75%) and sensitive to norfloxacin (96%). Ampicillin still sensitive for V. parahaemolyticus isolated from human stools. All of isolates were sensitive to namely chloramphenicol and floroquinolones (ciprofloxacin and norfloxacin agents). RAPD-PCR profiling with three primers (OPAR3, OPAR4 and OPAR8) produced four major clusters (R1, R2, R3 and R4), 7 minor clusters (I, II, III, IV, V, VI and VII) and three single isolates. Keywords: V. parahaemolyticus, MDR, RAPD 1. D. Ottaviani, I. Bacchiocchi, L. Masini, F. Leoni, A. Carraturo, M. Giammarioli, and G. Sbaraglia, Antimicrobial susceptibility of potentially halophilic vibrios isolated from seafood, International Journal of Antimicrobial Agents 18: 135-140, (2001).2. A. Cespedes, and E. Larson, Knowledge, attitude and practices regarding antibiotic use among Latinos in the United States: Review and Recommendations, American Journal of Infection Control 34: 495-502, (2006).3. M. Lesmana, D. Subekti, C.H. Simanjuntak, P. Tjaniadi, J. R. Campbell, and B. A. Ofoyo, Vibrio parahaemolyticus associated with cholera-like diarrhea among patients in North Jakarta, Indonesia, Diagnostic Microbiology and Infectious Disease, 39: 71-75, (2001).4. S. Lu, B. Liu, B. Zhou, And R. E. Levin, Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from two retail Sources and the Genetic Diversity of isolates as Determined by RAPD-PCR Analysis, Food Biotechnology, 20: 193-209, (2006).5. M. Nishibuchi, Vibrio parahaemolyticus. In International handbook of foodborne pathogens, ed. M.D. Milliots and J. W. Bier, United States: Marcel Dekker, Inc. P, 2004, 237-252.6. L. Poirel, M. R. Martinez, H. Mammeri, A. Liard, and P. Nordmann, Origin of Plasmid-Mediated Quinolone Resistance Determinant QnrA, Antimicrobial Agents and Chemotherapy, 49: 3523-3525, (2005).7. S. Radu, N. Elhadi, Z. Hassan, G. Rusul, S. Lihan, N. Fifadara, Yuherman and E. Purwati, Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): antimicrobial resistance, plasmid profiles and random amplification of polymorphic DNA analysis, FEMS Microbiology Letters, 165: 139–143, (1998).8. S. Radu, N. Ahmad, F. H. Ling, and A. Reezal, Prevalence and resistance to antibiotics for Aeromonas species from retail fish in Malaysia, International of Journal Food Microbiology, 81: 261–266, (2003).9. B. Sarkar, N. R. Chowdhury, G. B. Nair, M. Nishibuchi, S. Yamasaki, Y. Takeda, S. K. Gupta, S. K. Bhattacharya, and Ramamurthy, Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhea in Calcutta, India, World Journal of Microbiology and Biotechnology, 19: 771-776, (2003). 10. S. Schwarz, and E. Chaslus-Dancla, Use of antimicrobials in veterinary medicine and mechanisms of resistance, Veterinary Residue, 32: 201–225, (2001).11. H. Sörum, and T.M. L’Abèe-Lund,. Antibiotic resistance in food-related bacteria – a result of interfering with the global web of bacterial genetics, International Journal of Food Microbiology, 78: 43–56, (2002).12. P. Tjaniadi, M. Lesmana, D. Subekti, N. Machpud, S. Komalarini, W. Santoso, C. H. Simanjuntak, N. Punjabi, J. R. Campbell, W. K. Alexander, H. J. Beecham, A. L. Corwin, and B. A. Oyofo, Antimicrobial Resistance of Bacterial Pathogens Associated with Diarrheal Patients in Indonesia, American Journal of Tropical Medicine and Hygiene, 68: 666-670, (2003).13. X. Zhao, and D. Drlica, Restricting the Selection of Antibiotic-Resistant Mutants: A General Strategy Derived from Fluoroquinolone Studies, Clinical Infectious Diseases, 33: S147-S156, (2001).

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Bacteriocins, Antimicrobial Peptides from Bacterial Origin: Overview of Their Biology and Their Impact against Multidrug-Resistant Bacteria

Microorganisms ◽

10.3390/microorganisms8050639 ◽

2020 ◽

Vol 8 (5) ◽

pp. 639 ◽

Cited By ~ 13

Author(s):

Alexis Simons ◽

Kamel Alhanout ◽

Raphaël E. Duval

Keyword(s):

Antimicrobial Resistance ◽

Bacterial Infections ◽

Antimicrobial Agents ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Multidrug Resistant Bacteria ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Further Development

Currently, the emergence and ongoing dissemination of antimicrobial resistance among bacteria are critical health and economic issue, leading to increased rates of morbidity and mortality related to bacterial infections. Research and development for new antimicrobial agents is currently needed to overcome this problem. Among the different approaches studied, bacteriocins seem to be a promising possibility. These molecules are peptides naturally synthesized by ribosomes, produced by both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), which will allow these bacteriocin producers to survive in highly competitive polymicrobial environment. Bacteriocins exhibit antimicrobial activity with variable spectrum depending on the peptide, which may target several bacteria. Already used in some areas such as agro-food, bacteriocins may be considered as interesting candidates for further development as antimicrobial agents used in health contexts, particularly considering the issue of antimicrobial resistance. The aim of this review is to present an updated global report on the biology of bacteriocins produced by GPB and GNB, as well as their antibacterial activity against relevant bacterial pathogens, and especially against multidrug-resistant bacteria.

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Antimicrobial Resistance in the Intensive Care Unit

Journal of Intensive Care Medicine ◽

10.1177/0885066615619895 ◽

2016 ◽

Vol 32 (1) ◽

pp. 25-37 ◽

Cited By ~ 58

Author(s):

Shawn H. MacVane

Keyword(s):

Intensive Care ◽

Antimicrobial Resistance ◽

Bacterial Infections ◽

Antimicrobial Agents ◽

Treatment Options ◽

Hospital Costs ◽

High Morbidity ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Gram Negative Organisms

Bacterial infections are a frequent cause of hospitalization, and nosocomial infections are an increasingly common condition, particularly within the acute/critical care setting. Infection control practices and new antimicrobial development have primarily focused on gram-positive bacteria; however, in recent years, the incidence of infections caused by gram-negative bacteria has risen considerably in intensive care units. Infections caused by multidrug-resistant (MDR) gram-negative organisms are associated with high morbidity and mortality, with significant direct and indirect costs resulting from prolonged hospitalizations due to antibiotic treatment failures. Of particular concern is the increasing prevalence of antimicrobial resistance to β-lactam antibiotics (including carbapenems) among Pseudomonas aeruginosa and Acinetobacter baumannii and, recently, among pathogens of the Enterobacteriaceae family. Treatment options for infections caused by these pathogens are limited. Antimicrobial stewardship programs focus on optimizing the appropriate use of currently available antimicrobial agents with the goals of improving outcomes for patients with infections caused by MDR gram-negative organisms, slowing the progression of antimicrobial resistance, and reducing hospital costs. Newly approved treatment options are available, such as β-lactam/β-lactamase inhibitor combinations, which significantly extend the armamentarium against MDR gram-negative bacteria.

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Antimicrobial Resistance of Food-Related Salmonella Isolates, 1999–2000

Journal of Food Protection ◽

10.4315/0362-028x-65.4.603 ◽

2002 ◽

Vol 65 (4) ◽

pp. 603-608 ◽

Cited By ~ 36

Author(s):

CONNIE R. KIESSLING ◽

JEFFREY H. CUTTING ◽

MERCEDES LOFTIS ◽

WILLIAM M. KIESSLING ◽

ATIN R. DATTA ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Food Supply ◽

Antimicrobial Agents ◽

The United States ◽

Health Concern ◽

Fiscal Year ◽

Imported Food ◽

Foodborne Infection ◽

Year 2000 ◽

The U.S

Salmonellosis is a major foodborne infection in the United States, and strains of Salmonella that are resistant to a variety of antimicrobial agents have become a major public health concern. To estimate the incidence of antimicrobial-resistant Salmonella in our food supply, the U.S. Food and Drug Administration (FDA) has initiated screening of foodborne isolates for sensitivity to antimicrobial agents, including several antibiotics. Salmonella cultures (n = 502) isolated by FDA laboratories during fiscal year 2000 (1 October 1999 through 30 September 2000) from domestic and imported food products and related samples were tested for susceptibility to each of 12 antimicrobial agents using a disc diffusion assay. Because all isolates were resistant to rifampin (5 or 25 μg), only results with the remaining 11 antimicrobial agents are discussed in this paper. Of the 502 isolates, 247 (49.2%) were resistant to one or more antimicrobial agents, and of these 247 isolates, 170 (68.8%) were resistant to one antimicrobial agent, 33 (13.4%) to two antimicrobial agents, 25 (10.1%) to three antimicrobial agents, 7 (2.8%) to four antimicrobial agents, 8 (3.2%) to five antimicrobial agents, and 2 (0.8%) each to six and seven antimicrobial agents. No isolates were resistant to norfloxacin, whereas only seven were resistant to sulfamethoxazole/trimethoprim, six to trimethoprim, three to gentamicin, and one to ciprofloxacin. These results, for the first time, provide a baseline of data on the incidence of antimicrobial-resistant Salmonella in the U.S. food supply, which should be useful in determining the evolution of antimicrobial resistance in the future.

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Antimicrobial Resistance among Clinical Isolates ofStreptococcus pneumoniae in the United States during 1999–2000, Including a Comparison of Resistance Rates since 1994–1995

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.6.1721-1729.2001 ◽

2001 ◽

Vol 45 (6) ◽

pp. 1721-1729 ◽

Cited By ~ 390

Author(s):

Gary V. Doern ◽

Kristopher P. Heilmann ◽

Holly K. Huynh ◽

Paul R. Rhomberg ◽

Stacy L. Coffman ◽

...

Keyword(s):

United States ◽

Antimicrobial Resistance ◽

Antimicrobial Agents ◽

Rank Order ◽

The United States ◽

Clinical Isolates ◽

Beta Lactam ◽

High Level ◽

Resistance Rates

ABSTRACT A total of 1,531 recent clinical isolates of Streptococcus pneumoniae were collected from 33 medical centers nationwide during the winter of 1999–2000 and characterized at a central laboratory. Of these isolates, 34.2% were penicillin nonsusceptible (MIC ≥ 0.12 μg/ml) and 21.5% were high-level resistant (MIC ≥ 2 μg/ml). MICs to all beta-lactam antimicrobials increased as penicillin MICs increased. Resistance rates among non-beta-lactam agents were the following: macrolides, 25.2 to 25.7%; clindamycin, 8.9%; tetracycline, 16.3%; chloramphenicol, 8.3%; and trimethoprim-sulfamethoxazole (TMP-SMX), 30.3%. Resistance to non-beta-lactam agents was higher among penicillin-resistant strains than penicillin-susceptible strains; 22.4% of S. pneumoniae were multiresistant. Resistance to vancomycin and quinupristin-dalfopristin was not detected. Resistance to rifampin was 0.1%. Testing of seven fluoroquinolones resulted in the following rank order of in vitro activity: gemifloxacin > sitafloxacin > moxifloxacin > gatifloxacin > levofloxacin = ciprofloxacin > ofloxacin. For 1.4% of strains, ciprofloxacin MICs were ≥4 μg/ml. The MIC90s (MICs at which 90% of isolates were inhibited) of two ketolides were 0.06 μg/ml (ABT773) and 0.12 μg/ml (telithromycin). The MIC90 of linezolid was 2 μg/ml. Overall, antimicrobial resistance was highest among middle ear fluid and sinus isolates of S. pneumoniae; lowest resistance rates were noted with isolates from cerebrospinal fluid and blood. Resistant isolates were most often recovered from children 0 to 5 years of age and from patients in the southeastern United States. This study represents a continuation of two previous national studies, one in 1994–1995 and the other in 1997–1998. Resistance rates with S. pneumoniae have increased markedly in the United States during the past 5 years. Increases in resistance from 1994–1995 to 1999–2000 for selected antimicrobial agents were as follows: penicillin, 10.6%; erythromycin, 16.1%; tetracycline, 9.0%; TMP-SMX, 9.1%; and chloramphenicol, 4.0%, the increase in multiresistance was 13.3%. Despite awareness and prevention efforts, antimicrobial resistance with S. pneumoniae continues to increase in the United States.

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