Antibiotic resistance : causes and risk factors, mechanisms

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Antibiotic resistance : causes and risk factors, mechanisms

PHARMACOLOGY – RESEARCH, SAFETY TESTING AND REGULATION SERIES

ANTIBIOTIC RESISTANCE:

CAUSES AND RISK FACTORS,

MECHANISMS AND ALTERNATIVES

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PHARMACOLOGY – RESEARCH, SAFETY

TESTING AND REGULATION SERIES

Poisons: Physiologically Active Substances

S. B. Zotov and O. I. Tuzhikov

ISBN: 978-1-60741-973-0

Antibiotic Resistance: Causes and Risk Factors,

Mechanisms and Alternatives

Adriel R. Bonilla and Kaden P. Muniz (Editors)

2009 ISBN: 978-1-60741-623-4


PHARMACOLOGY – RESEARCH, SAFETY TESTING AND REGULATION SERIES

ANTIBIOTIC RESISTANCE:

CAUSES AND RISK FACTORS,

MECHANISMS AND ALTERNATIVES

ADRIEL R. BONILLA

AND

KADEN P. MUNIZ

EDITORS

Nova Science Publishers, Inc.

New York


Copyright © 2009 by Nova Science Publishers, Inc.

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Library of Congress Cataloging-in-Publication Data

Antibiotic resistance : causes and risk factors, mechanisms and alternatives / [edited by] Adriel R. Bonilla

and Kaden P. Muniz.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-1-61668-162-3 (E-Book)

1. Drug resistance in microorganisms. I. Bonilla, Adriel R. II. Muniz, Kaden P.

[DNLM: 1. Drug Resistance, Bacterial. 2. Anti-Bacterial Agents--therapeutic use. QW 52 A6287 2009]

QR177.A55 2009

616.9'041--dc22

2009024616

Published by Nova Science Publishers, Inc. New York


Contents

Preface

Chapter I

Chapter II

Chapter III

Chapter IV

Chapter V

Chapter VI

Chapter VII

The Phenomenon of Antibiotic Resistance

in an Evolutionary Perspective 1

Roberto Manfredi

Antibiotic Resistance and Probiotics: Roles,

Mechanisms and Evidence 5

Min-Tze Liong, Siok-Koon Yeo, Chiu-Yin Kuan,

Wai-Yee Fung and Joo-Ann Ewe

When Good Bugs Fight Bad – Designing Probiotics

to Control Antibiotic Resistant “Superbugs” 39

Roy D. Sleator

Modification of the Bacterial Phenotype as an Approach

to Counter the Emergence of Multidrug-Resistant Pathogens 43

Peter W. Taylor, Patricia Bernal and Andrea Zelmer

Performance to Antibiotics in Staphylococcus aureus,

Streptococcus agalactiae and Enterococcus spp. 79

Antonio Sorlózano, José Gutiérrez, Eva Román,

Juan de Dios Luna, Juan Román, Estrella Martín, José Liébana,

José Luis García, Samuel Bernal and Pilar Morales

Microbiological and Pharmacological Principles of Recent

Antibiotic Therapy 97

Cristina Carrasco, Antonio Sorlózano, José Cabeza,

Gonzalo Piédrola and José Gutiérrez

Bacteriophage Therapies and Enzybiotics: Novel Solutions

to Antibiotic Resistance 187

Noémie Manuelle Dorval Courchesne,

Albert Parisien and Christopher Q. Lan

vii


vi

Contents

Chapter VIII

Application of Computational Methods to Study Antibiotic

Resistance Mechanisms: Fluoroquinolone Resistance

as a Case Study 215

Sergio Madurga, Jordi Vila and Ernest Giralt

Chapter IX Energetics of MDR Microbial Cells 247

Waché Yves

Chapter X

Low Impact of OmpC and OmpF on Susceptibility to Antibiotics

in Clinical Isolates of Escherichia coli Producers of Extended-

Spectrum Beta-Lactamases 261

A. Sorlózano, A. Salmerón, F. Martínez-Checa, E. Villegas

J. D. Luna and J. Gutiérrez

Chapter XI Antibiotic Resistance in Mariculture Environments of China 271

Jingyi Zhao and Hongyue Dang

Chapter XII

Chapter XIII

Chapter XIV

Chapter XV

Antibiotic Resistance as a Method for Determining Sources

of Fecal Pollution in Water 283

Alexandria K. Graves and Charles Hagedorn

Genetic Regulation, Physiology, Assessment and Inhibition

of Efflux Pumps Responsible for Multi-Drug Resistant

Phenotypes of Bacterial Pathogens 313

L. Amaral, S. Fanning, G. Spengler, L. Rodrigues, C. Iversen,

M. Martins, A. Martins, M. Viveiros and I. Couto

Genetic and Phenotypic Characterization of Drug-Resistant

Mycobacterium Tuberculosis Isolates 333

Maysaa El Sayed Zaki

Tracking Down a New Putative Drug Target for Osteoporosis:

Structure of an Essential Region of the Proton Translocation

Channel of H + -V O -ATPase 345

Afonso M. S. Duarte and Marcus A. Hemminga

Chapter XVI Antibiotic Resistance in Food-Associated Lactic Acid Bacteria 371

Giorgio Giraffa

Chapter XVII Current Status of Antibiotic Resistance in Lactic Acid Bacteria 379

Leon M. T. Dicks, Svetoslav D. Todorov

and Bernadette D. G. M. Franco

Chapter XVIII Nanobiotics to Combat Bacterial Drug Resistance 425

Sampath C. Abeylath and Edward Turos

Chapter XIX

Veterinary Antibiotics and Their Possible Impact on Resistant

Bacteria in the Environment 467

Nicole Kemper

Index 497


Preface

Antibiotic resistance is the ability of a microorganism to withstand the effects of

antibiotics. There are three mechanisms that can cause antibiotic resistance: prevention of

interaction of drug with target, decreased uptake due to either an increased efflux or a

decreased influx of the antimicrobial agent and enzymatic modification or destruction of the

compound. In the past couple years, antibiotic resistance has become an increasing public

health concern. Tuberculosis, gonorrhea, malaria and childhood ear infections are just a few

of the diseases that have become hard to treat with antibiotic drugs. This book addresses the

concern that over the past few years, there has been a major rise in resistance to antibiotics

among gram-negative bacteria. New antibacterial drugs with novel modes of actions are

urgently required in order to fight against infection. Novel antibiotics such as linezolid,

carbapenem ertapenem, daptomycin and gemifloxacin are examined in this book. The genetic

approaches used in risk assessment of antibiotic resistance dissemination are looked at as

well. Furthermore, this book discusses the present studies on the use of veterinary antibiotics

in agriculture, on the occurrence of antibiotic compounds and resistant bacteria in soil and

water and clearly demonstrates the need for further studies.

Chapter I- The extraordinary capacity of a number of bacterial organisms to develop

resistance to antibiotic compounds remains a hot topic on the minds of doctors, hospital staff,

reporters, and the community since several years. It is also heralded as a textbook example of

“Darwinian evolution” continuously in action. As a consequence, these multiresistant

bacteria are being studied also by “evolutionary” scientists, with the hope that they will

reveal secrets or trace a pathway as to how molecules-to-man evolution could have happened.

Chapter II - Probiotics are live microorganisms that could confer health benefits on the

host once administered in adequate amounts. One of the main benefits of probiotic organisms

is their ability to improve gastrointestinal health, where they act as an important biodefense in

preventing colonization and subsequent proliferation of pathogenic bacteria in the intestines.

Additionally, they have also been used to treat and/or prevent antibiotic resistance in the host

via indirect mechanisms. Various studies have shown that probiotics could modulate the

immune system of the host leading to a reduced use of antibiotics, and could suppress the

growth of pathogenic bacteria thus limiting their transfer of resistant genes to the host.

A long history of safety has contributed to the acceptance of probiotics as a safe food

adjunct. However, recent studies have reported the occurrence of antibiotic resistance in


viii

Adriel R. Bonilla and Kaden P. Muniz

probiotics. Probiotics have also been found to translocate and were isolated from infected

lesions such as bacterial endocarditis and blood stream infections. These have led to the need

for further assessment of probiotics so that their negative consequences do not outweigh their

benefits. Antibiotic resistance and the ability to translocate of probiotics are now emphasized

and are suggested to be made as criteria for safety assessment and labeling prior to their use

as food supplements.

Much attention has been given to the factors and mechanisms involved on the acquired

antibiotic resistance of probiotics. Some studies have found that the species and origins of the

strains could affect the acquiring of antibiotic resistance, while others have demonstrated that

genes coding for antibiotic resistance are transferable among bacteria of different origins.

Recent laboratory advances have provided more details and information on some postulated

mechanisms. Probiotics could be antibiotic resistant inherently or acquired via vertical

evolution such as inactivation of enzymes, alteration of membrane protein channels and

efflux pumps, and/or via horizontal evolution of gene exchange.

Although antibiotic resistance of probiotics is rarely life threatening, it may serve as a

marker of more serious underlying diseases. This review will also present some in-vitro and

in-vivo evidence of the detections and isolation of antibiotic resistant probiotics,

epidemiology of strains found, and their surveillance, monitoring and disposal once detected.

There are also efforts to evaluate the need for elimination of antibiotic resistance although

few probiotics meant for human consumption are classified as resistant. Past reports have

found that a failure to demonstrate in-vitro horizontal gene transfer could not be used as a

yardstick to exclude the risk of dissemination of genes. It has been suggested that genetic

approaches are needed to eliminate antibiotic resistance, one being selective removal of

plasmids coding for antibiotic resistance. However, the issues of phenotypical and

genotypical alteration of the strain arise and that the impact of the genetic changes on the

probiotic properties remains unknown. The present review will also highlight the genetic

approaches used in risk assessment of antibiotic resistance dissemination.

Chapter III - An ever increasing dependence on conventional therapeutics coupled with

an alarming escalation in antibiotic resistance has facilitated the emergence of a new faction

of bacterial adversaries, the antibiotic resistant ‘superbugs’, the aetiological agents of

nosocomial infections. Against this backdrop scientists and clinicians are now struggling to

find viable therapeutic alternatives to antibiotics. One such alternative involves the use of

probiotics; live microorganisms, which when consumed in adequate amounts, confer a health

benefit to the host. Furthermore, a new generation of probiotics termed ‘designer probiotics’

have been engineered to target and neutralise specific bacterial pathogens, viruses and toxins

while at the same time persisting for longer periods in foods and other delivery vehicles prior

to ingestion and subsequently within the gastrointestinal tract.

Chapter IV - Antibiotics are among the most beneficial drugs ever introduced but their

utility has been compromised by the emergence and spread of antibiotic resistant bacteria.

Currently useful antibiotics impose enormous selective pressure on bacterial populations and

the use and overuse of these agents has led to the evolution, in a relatively short timeframe, of

multidrug-resistant pathogens capable of rapid and efficient horizontal transmission of genes

encoding resistance determinants. New antibacterial drugs with novel modes of action are

urgently required in order to continue the fight against infection: unfortunately, the upturn in


Preface

ix

the incidence of resistance has coincided with a marked reduction by the pharmaceutical

industry in efforts to develop new agents. Even “professional” pathogens such as

Staphylococcus aureus, a bacterium equipped with an impressive array of virulence effectors

for damaging the host, require a reduction in the host’s defenses in order to colonize, enter

tissue and cause infection. Correction of this imbalance between host and pathogen may

provide a novel approach to the therapy of severe bacterial infections, by either shoring up

immune defenses or modifying the bacteria at the site of infection to render them less able to

survive in the host. Thus, pathogens may survive, multiply and cause infection because they

are virulent or resistant to antibiotics; therapeutic agents that modify these properties in vivo

may resolve infections, either alone or in combination with conventional antibiotic

chemotherapy, in a way that may not readily lead to drug resistant bacterial survivors.

Examples of such modifiers of the bacterial phenotype, together with other emerging

approaches to the treatment of bacterial infections, are described.

Chapter V - Staphylococcus aureus, Streptococcus agalactiae and Enterococcus spp. are

bacteria with a high capacity for acquiring determinants of resistance to antibiotics, giving

rise to multi-resistance problems. It is assumed that this is related to consumption of

antibiotics. The study objectives were to determine the antibiotic susceptibility of these three

species in two Spanish hospitals and establish the relationship between consumption of

antibiotics and their activity. The activity of antibiotics was evaluated against 1000 clinical

isolates of S. aureus, Enterococcus spp. and S. agalactiae identified in the Microbiology

Services of San Cecilio Hospital in Granada and Valme Hospital in Seville, determining the

relationship between the consumption of antibiotics and susceptibility findings.

Glycopeptides and linezolid were the only antibiotics active against 100% of clinical isolates

of S. aureus, with cotrimoxazole and rifampicin also showing excellent activity. All isolates

of S. agalactiae were susceptible to ampicillin, cefotaxime, vancomycin and linezolid, with

an excellent activity for levofloxacin. All enterococci were susceptible to ampicillin,

glycopeptides and linezolid. Finally, a statistically significant inverse relationship was found

for the whole set of antibiotics between their consumption and the susceptibility to them of S.

aureus. The three species retained susceptibility to glycopeptides and linezolid. A good

susceptibility was also shown by S. aureus to cotrimoxazole and rifampicin and by

enterococci and S. agalactiae to ampicillin. There was a significant association between

antibiotic consumption and rates of resistance to S. aureus.

Chapter VI - The past few years have seen a major rise in resistance to antibiotics among

Gram-negative bacteria (beta-lactamase [ESBL]-producing enterobacteria, carbapenemresistant

P. aeruginosa and A. baumannii …) and, especially, among Gram-positive bacteria

(methicillin-resistant S. aureus [MRSA], glycopeptide-intermediate [GISA] or resistant S.

aureus [GRSA], vancomycin-resistant enterococci [VRE], multi-drug resistant S.

pneumoniae [MDRSP]). This trend has considerably reduced therapeutic options and brought

about a need for novel antibiotics, which have been approved by the Food and Drugs

Administration (FDA) since 2000.

The first of these was linezolid, an oxazolidinone for the treatment of nosocomial and

community-acquired pneumonias and skin and soft tissue infections caused by Gram-positive

bacteria, including MRSA, GRSA, VRE, and MDRSP. The cephalosporin cefditoren pivoxil

was approved to treat community-acquired respiratory infections (upper and lower airways)


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Adriel R. Bonilla and Kaden P. Muniz

and uncomplicated infections of skin and soft tissues produced by Gram-positive or -negative

bacteria that are not resistant to this group of antibiotics.

The carbapenem ertapenem is indicated for the treatment of intra-abdominal, skin and

soft tissue infections, and community acquired pneumonia caused by Gram-positive ornegative

microorganisms and is especially indicated for infections produced by ESBLproducing

enterobacteria. Doripenem is a new carbapenem that can be used against intraabdominal

and urinary infections due to Gram-negative or -positive microorganisms at a

lower dose than required for other antibiotics in this group.

Daptomycin is a useful antibiotic for monotherapy of bacteremia and skin and soft tissue

infections caused by Gram-positive multiresistant bacteria (MRSA, GISA, GRSA).

Gemifloxacin is a new quinolone for treating respiratory infections (e.g., community

pneumonia or bronchitis) caused by Gram-positive and –negative bacteria, including

MDRSP. Telithromycin offers an alternative treatment for community-acquired pneumonia

due to Gram-positive and –negative microorganisms, including MDRSP.

Tigecycline was approved for intra-abdominal, skin, and soft tissue infections caused by

Gram-positive and–negative bacteria, including multi-resistant bacteria (ESBL, MRSA).

Retapamulin is used topically for impetigo produced by Gram-positive

microorganisms.The objective of our study will be to review the in vitro antibiotic activity,

action mechanisms, and resistance (if reported) of the above antibiotics and to describe their

pharmacokinetic and pharmacodynamic properties.

Chapter VII - Bacteriophages (phages) and bacterial cell wall hydrolases (BCWHs) have

been recognized as promising alternative antibacterials. Phages are bacterial virsuses that kill

bacteria by causing bacteriolysis. It is estimated that there are about 10 31 phages on earth and

approximately 5,100 have been identified and reported towards the end of last century,

providing a vast pool of candidates for applications such as phage therapy. Phages are highly

effective and specific to target bacteria. They have been generally accepted as effective

antibacterials that could potentially provide safe and cost effective means for the treatment

and prophylaxis of bacterial infections. BCWHs are lytic enzymes that cause bacteriolysis by

hydrolyzing the peptidoglycan of bacterial cell wall. BCWHs are also called enzybiotics

because they are enzymes that have antibiotic-like antibacterial activities. They are produced

by amost all cellular organisms including microorganisms, plants, and animals. Virolysins

(also called lysins, endolysins) are a special group of BCWHs encoded by phages but

produced naturally by bacteria infected by lytic phages. They are probably the most

promising enzybiotics.

Chapter VIII - Antibiotic resistance can occur via three mechanisms: prevention of

interaction of the drug with target; decreased uptake due to either an increased efflux or a

decreased influx of the antimicrobial agent and enzymatic modification or destruction of the

compound. Herein we review the results from computational studies on said mechanisms. For

the first mechanism, the bacteria produce mutant versions of antibiotic targets. This process

has been studied using several levels of theory, from costly quantum mechanics calculations

to classical molecular dynamics calculations. For the decreased uptake mechanisms, since the

atomic structure of several influx pores and efflux pumps were resolved recently, homology

modelling studies and large molecular dynamics simulations allow us to understand this

resistance mechanism at the molecular level. Computational studies with antibiotic molecules


Preface

xi

(mainly ß-lactam compounds) studying the resistance mechanism in which the antibiotic

compound is transformed will be also summarized. ß-lactamases are bacterial enzymes

responsible for most of the resistance against ß-lactam antibiotics. These defensive enzymes,

prevalent in nearly every pathogenic bacterial strain, hydrolyze the ß-lactam ring and release

the cleaved, inactive antibiotics. Computational studies on this reaction provided us with

knowledge on the molecular mechanism of action of these enzymes. The results may prove

valuable in the design of new antibiotic derivatives with improved activity in resistant strains.

Finally, a case study on the mechanism of resistance of fluoroquinolones is explained in

detail. Extensive use and misuse of fluoroquinolones in human and veterinary medicine has

led to the emergence and spread of resistant clones. These have appeared mainly through

mutations in the structural genes that encode their intracellular targets (DNA gyrase and

topoisomerase IV) as well as via modifications in membrane permeability (e.g. decreased

influx and/or increased efflux). We analyzed bacterial resistance to fluoroquinolones that

arises from mutations in the DNA gyrase target protein. We studied the binding mode of

ciprofloxacin, levofloxacin, and moxifloxacin to DNA gyrase by docking calculations. The

binding model obtained enabled us to study, at the atomic level, the resistance mechanism

associated with the most common gyrA mutations in E. coli fluoroquinolone-resistant strains.

Chapter IX - Multidrug efflux pumps are important in microbial resistance to antibiotics.

The energetics of some systems has been characterised in vitro but the impact on whole cell

energetics is less studied. In this chapter, after the rapid presentation of the different MDR

efflux families, the energetics of some MDR transporters will be described. The impact of

these transporters on cell energetics is discussed and the difficulties in studying MDR efflux

energetics in whole cell systems will be presented with a description of available techniques.

The importance of this field of investigation will be demonstrated with an example of a

microbial global strategy to resist to a cationic compound.

Chapter X - The activity of 12 antibiotics in Mueller-Hinton (MH) broth and Nutrient-

Broth (NB) was assessed by microdilution in 50 clinical isolates of Escherichia coli

producers of extended-spectrum beta-lactamases (ESBLs). In addition, the expression pattern

of OmpC and OmpF porins was analyzed after culture of isolates in the two media. Three

porin expression patterns were obtained: 13 isolates expressed both porins in both media, 8

expressed one porin in both media, and 29 expressed both porins in NB and one in MH.

Analysis of the relationship between modification of porin expression and MIC values of the

different antibiotics tested was unable to demonstrate, using this methodology, that porin loss

was responsible for clinically relevant changes in the MIC of these clinical isolates.

Chapter XI - China has become the largest mariculture country of the world. The

extensive and intense use of antibiotics in mariculture for the prevention and treatment of

microbial infections of the farmed marine animals exerts a selective pressure on

environmental bacteria. The mariculture environment may have become a reservoir and

dissemination source of antibiotic resistance. The prevalence of antibiotic resistance may

undermine the effectiveness of various antibiotics, alter the natural microbiota, disturb the

ecological equilibria, and create a potential threat to human health and welfare. The

emergence of antibiotic resistant bacteria in mariculture and related environments has

become a great concern of environmental and human health. There is an urgent need to

develop a monitoring platform to investigate the antibiotic resistant bacteria and their


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Adriel R. Bonilla and Kaden P. Muniz

resistance mechanisms in mariculture environments, for effective prevention and control of

the spread and evolution of harmful antibiotic resistant bacteria and their resistance

determinants. Certain research effort has been made to evaluate the abundance and diversity

of the antibiotic-resistant microbes and their resistance genes in China mariculture

environments. A preliminary review on this issue is provided in this paper. As the biggest

mariculture country, China will take the responsibility and resolution for the control and

elimination of antibiotic resistance in mariculture environment.

Chapter XII - Understanding the origin of fecal pollution is essential in assessing

potential health risks as well as for determining the actions necessary to remediate the

problem of waters contaminated by fecal matter. As a result, microbial source tracking (MST)

methods aimed at determining sources of fecal pollution have evolved over the past decade.

Antibiotic resistance analysis (ARA) has emerged as one MST tool that has proven useful for

determining the sources of fecal pollution in environmental waters.

Antibiotic resistance analysis is a simple, rapid, and inexpensive method for performing

MST. It is a published method that has been independently validated by a variety of tests

designed to improve library-based methods, and is currently being used in laboratories across

the United States as well as internationally.

The ARA method is based on the rationale that antibiotics exert selective pressure on the

fecal flora of the animals that ingest antibiotics. Different types of animals receive differential

exposure to antibiotics and as a result, different animals will have bacteria with at least

reasonably source-specific patterns of antibiotic resistance. The ARA procedure involves the

isolation and culturing of a target organism, (usually Escherichia coli or Enterococcus sp.),

then replica plating the isolates on media containing various antibiotics at pre-selected

concentrations. After the plates are incubated, the organisms are scored according to their

resistance to the antibiotics to generate an antibiotic resistance profile (phenotypic

fingerprint). These fingerprints are then evaluated by a multivariate statistical method such as

discriminate analysis (DA).

Discriminant analysis classifies the bacteria based on shared patterns of antibiotic

resistance, and the results are pooled to form a “known source database” (or library) of

antibiotic resistance patterns from different fecal sources. The known source database is

summarized in the form of a classification table. The average rate of correct classification is

the average rate that known source isolates are correctly classified, and is used to measure the

reliability of the library. Additional method performance criteria to ensure the reliability of

the database are also used, including library decloning, construction of larger libraries

representing more diverse host populations, and realistic tests of method accuracy, such as

incorporating independently collected proficiency isolates and samples. Isolates recovered

from water samples are compared to the known source database to identify an isolate as being

either human or animal derived.

Antibiotic resistance analysis is an MST tool that is most appropriate for small, relatively

simple rural watersheds. The application of ARA to large-scale complex systems is more

problematic as the wider genetic variability present in target organism populations impacts

the phenotypic expression of antibiotic resistance. The potential of this genetic diversity

occurring in many different host sources leads to the concern of not being able to fully

capture that diversity in a known source library, with subsequent reductions in classification


Preface

xiii

accuracy of water isolates (unknown sources). This is particularly relevant when multiple

sources of pollution are contributing to watershed contamination at comparatively similar

levels. However, the method performance issues that must be addressed with ARA (and are

described in this chapter) are identical for any library-based approach and can also be

modified to evaluate library-independent MST methods as well.

This chapter describes the advantages and disadvantages of ARA, presents case studies

where the method has been used in a variety of different environmental situations, responds

to criticisms of ARA, and details six method performance criteria that should be applied to

every MST method. Such evaluations are long overdue in the rapidly developing field of

MST.

Chapter XIII - The response of bacteria to a given antibiotic or other toxic agent that

does not immediately kill them results in an adaptive response that secures their survival.

This response may involve a number of distinct mechanisms, among which is the activation

of genes that promote the appearance of transporters that extrude the agent prior to its

reaching its target. These transporters extrude a large variety of chemically unrelated

compounds and hence they bestow on the bacterium a multi-drug resistant (MDR) phenotype.

The appearance of this MDR phenotype during therapy makes therapy problematic.

Understanding the genetic and physiological properties of MDR efflux pumps is an absolute

requirement if MDR bacterial infections are to be successfully managed. There is much to

learn and methods that afford the needed understanding will eventually pave the way for the

successful management of an MDR bacterial infection. Therefore, the application of recently

developed methods that assess the MDR bacterium at its genetic and physiological levels is

the focus of this chapter. The material presented in this chapter is only the beginning for the

evaluation and assessment of MDR efflux pumps.

Chapter XIV - The reemergence of Mycobacterium tuberculosis with increasing numbers

of multi-drug resistant (MDR) strains has increased the need for rapid diagnostic methods.

Molecular bases of drug resistance have been identified for all of the main

antituberculous drugs, and drug resistance results from changes in several target genes, some

of which are still undefined. Drug resistance in M. tuberculosis is due to the acquisition of

mutations in chromosomally encoded genes and the generation of multidrug resistance is a

consequence of serial accumulation of mutations primarily due to inadequate therapy. Several

studies have shown that resistance to isoniazid (INTI) is due to mutaions in kat G gene. The

rpo B gene, which encodes the subunit of RNA polymerase, harbors a mutation in an 81 bp

region in about 95% of rifampicin (RIF) reistant M. tuberculosis strains recovered globally.

Streptomycin (STR) resistance is due to mutations in rrs and rpsl genes which encodes 16S

SrRNA and ribosomal protein S12 respectively. Approximately 65% of clinical isolates

resistant to ethambutol have a mutation in the embB gene.

Several susceptibility phenotypes methods have been developed. Among them were

radiometric systems such as the BACTEC460 TB system reduces the test time considerably,

but they still labor intensive, expensive and require manipulation of radioactive substances.

Rapid promising approach to determine drug resistant strains especially for rifampicin

(RIF) is the use of mycobacteriophage. A number of low-cost colorimetric AST assays, such

as the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the

Alamar blue assay, and an assay based on microscopic detection of cord-like growth by M.


xiv

Adriel R. Bonilla and Kaden P. Muniz

tuberculosis, have been described. However, these tests have limitations; mycobacteria other

than M. tuberculosis can produce cord factor, and INH can interfere with the formazan

production in the MTT assay and give rise to false resistant results. Moreover, the use of a

liquid medium in a microtiter plate format in these tests may be disadvantageous not only as a

biohazard but also due to possible Contamination between wells.

The goal of the present study is to characterize 100 drug-resistant Mycobacterium

tuberculosis (MTB) isolates with respect to their drug susceptibility phenotypes to four

common anti-tuberculosis drugs (rifampicin, ethambutol, isoniazid andstreptomycin) and the

relationship between such phenotypes and the patterns of genetic mutations in the

corresponding resistance genes ( rpoB, embB, katG, Rpsl) METHODS: The MIC values of

the aforementioned anti-tuberculosis drugs were determined for each of the 100 drugresistant

MTB clinical isolates by the absolute concentration method and by BACTEC460).

Genetic mutations in the corresponding resistance genes in these MTB isolates were

identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer

conformation analysis (SSCP/MPAC).

Phenotypic resistance was found in all isolates. All isolates were resistant to ethambutol,

70% isolates were resistant to isoniazid, 50% isolates were resistant to streptomycin and 20%

of the isolates were resistant to rifampicin. Genotypic mutations in the studied resistance to

rifampicin, isoniazid, streptomycin and ethambutol were 20%, 70%, 80%, and 100%

respectively.

These findings expand the spectrum of potential resistance-related mutations in MTB

clinical isolates and help consolidate the framework for the development of molecular

methods for delineating the drug susceptibility profiles of MTB isolates in clinical

laboratories.

Chapter XV - In the last decades, osteoporosis has become a major subject in the field of

drug discovery and design. One of the proteins recently considered relevant to use as a drug

target is the membrane-bound enzyme H + -V O -ATPase. This proton pump is located in the

osteoclast cells, which are positioned at the bone surface. In these cells the enzyme controls

the proton flux to the bone and consequently bone resorption. One major task on drug design

is the knowledge of the secondary and tertiary structure of the enzyme involved. The

topology of the V-ATPase protein complex has been largely established, however, the threedimensional

structure is only known for some individual subunits. This chapter reviews our

recently published work on the secondary and tertiary structure of the proton translocation

channel located in subunit a of the V-ATPase complex. For this purpose, we designed two

peptides consisting of 25 and 37 amino acid residues, representing the seventh

transmembrane segment of subunit a, which encompass the proton translocation channel as

well as the region of interest for interaction with possible inhibitors. Using a combination of

NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy the structure of

these V-ATPase peptides was studied in different membrane-mimicking environments. The

results indicate that a primordial transmembrane segment of this protein region adopts an -

helical conformation and is longer than proposed by topological studies. Moreover, this

segment exhibits a hinge region located near the cytoplasmic end of the channel. It is

proposed that the presence of this hinge allows the opening and closing of the proton

translocation channel and provides flexibility for the channel to act as a binding pocket for


Preface

xv

inhibitors. These findings can be used as tools in the design of new drugs that can control the

activity of V-ATPase in osteoclast cells, helping in this way the fight against osteoporosis.

Chapter XVI - The emergence of antimicrobial-resistant microorganisms in both humans

and livestock is a growing concern. In recent years, increased focus has been given to food as

a carrier of antibiotic resistance (AR) genes. However, there have been few systematic

studies to investigate acquired AR in lactic acid bacteria (LAB), which constitute common

components of the microbial community of food and play a relevant role in food

fermentation. In Europe, the Food Safety Agency (EFSA) received a request to assess the

safety of microorganisms throughout the food chain and proposed the Qualified Presumption

of Safety (QPS) approach. In the QPS approach, safety assessment will depend upon the body

of knowledge available for a given microorganism. Apparently, there are no specific concerns

regarding most of the food-associated LAB species, which have a long history of safe use in

the food chain, provided that the lack of acquired AR is systematically demonstrated.

Consequently, increased efforts have been devoted in recent years to gain more insight into

the diffusion of AR phenotypes within food-associated LAB, with particular emphasis on

those applied as starter cultures or probiotics. Presently available literature data support the

view that, in antibiotic-challenged habitats, LAB (especially enterococci) like other bacteria

are involved in the transfer of resistance traits over species and genus borders, with important

safety implications. The prevalence of such bacteria with acquired genetically-exchangeable

resistances is high in animals and humans that are regularly treated with antibiotics. This led

to the European ban of the antibiotics used as growth promoters in animal feed. Such

measures should, however, be complemented by a more prudent use of antibiotics in both

human and animal clinical therapy.

Chapter XVII - Lactic acid bacteria (LAB) consist of the genera Lactobacillus,

Carnobacterium, Lactococcus, Enterococcus, Streptococcus, Leuconostoc, Pediococcus,

Desemzia, Isobacilum, Paralactobacillus, Tetragenococcus, Trichococcus, Weissella,

Oenococcus and Melissococcus and are present in many different habitats, including food and

the human and animal intestinal tract. The long history of LAB with fermented foods have

bestowed on them GRAS (generally regarded as safe) status. Intestinal strains play an

important role in ensuring a healthy microbial balance. Their presence may lead to increased

cytokine production and they may even play a role in preventing the growth of carcinogenic

cells. A few rare cases of excessive immune stimulation, systemic infections, arthritis and the

formation of hepatobiliary lesions have been reported for some strains of LAB. Such

symptoms are usually associated with strains penetrating the mucus layer and epithelial cells.

Lactic acid bacteria in food and the gastro-intestinal (GI) tract could act as a potential

reservoir of antibiotic resistance genes and may participate in the exchange of genes with

strains present in same environment to produce multidrug resistant strains. Lactobacillus,

Pediococcus and Leuconostoc spp. have a high natural resistance to vancomycin. Some

Lactobacillus spp. are by nature resistant to bacitracin, cefoxitin, ciprofloxacin, fusidic acid,

kanamycin, gentamycin, metronidazole, nitrofurantoin, norfloxacin, streptomycin,

sulphadiazine, teicoplanin, trimethoprim/sulphamethoxazole, and vancomycin. A few cases

of Enterococcus infection have been associated with abnormal physiological conditions,

underlying disease and immunosuppression. Enterococcus faecalis is the most dominant

species in general GI infections, whereas Enterococcus faecium may cause enterococcal


xvi

Adriel R. Bonilla and Kaden P. Muniz

bacteremia. Enterococci have intrinsic resistance to cephalosporins, beta-lactams,

sulphonamides and low levels of clindamycin and aminoglycosides. Resistance to

chloramphenicol, erythromycin, high levels of clindamycin, aminoglycosides, tetracycline, -

lactams (-lactamase or penicillinase), fluoroquinolones and glycopeptides is usually

acquired. Vancomycin-resistant enterococci (VRE) with vanA, vanB, vanC1, vanC2, vanC3,

vanD and vanE genes have led to a number of nosocomial infections. VanA-type strains

confer high level inducible resistance to vancomycin and teicoplanin, whereas VanB-type

strains display variable levels of inducible resistance. VRE are highly resistant to most

antibiotics and successful treatment is a major problem. The aim of this review is to discuss

antibiotic resistance amongst LAB and highlight the genetic determinants involved in

horizontal and vertical transfer of resistance genes.

Chapter XVIII - One of the major medical advances of the 20th century has been the

development of effective antibiotics which have had a profound impact on the quality of

human life. The ability to treat and cure deadly infections and bacterial diseases has forever

changed our medical profession and way of life, providing unprecedented relief from pain,

suffering, and death due to microbial infection. However, consistent overuse and misuse of

powerful broad-spectrum antibiotics over several decades has added to and indeed

accelerated the spread of microbial drug resistance. Resistance mechanisms have been found

for every class of antibiotic agent used clinically. A major global healthcare problem relates

to serious infections caused by bacteria having resistance to commonly used antibiotics. Not

only are such infections typically more severe than those of antibiotic-responsive ones, they

also require more aggressive interventive measures and are thus significantly more difficult

and expensive to treat. Consequently, as we enter the 21 st century, a pressing need exists for

novel approaches to effectively deal with drug-resistant microbes and the health problems

they pose. Nature may provide answers to help guide us, in the form of viral warriors known

as bacteriophages, as a means to control and subdue pathogenic bacteria in their natural

habitat. Like phages, which emerged presumably through a long evolutionary process,

researchers now are acquiring the capabilities to target and neutralize deadly bacteria with

nanoparticle-based antibiotics, or nanobiotics. This chapter discusses the different types of

nanobiotics being investigated for delivering and improving therapeutic efficacy of a wide

assortment of anti-infectives. The emphasis will be on nanoparticle containers or vehicles to

overcome bacterial drug resistance that in essence protect and deliver various classes of

antibiotic drugs, including -lactams, fluoroquinolones, gentamycin, and vancomycin. A

brief synopsis is provided on the role nanobiotics could play in drug discovery and

development.

Chapter XIX - In this chapter, recent studies on the fate of antibiotics, and especially

antibiotics used in animal husbandry, are evaluated under the aspect of potential risks for

human health. Because the assumed quantity of antibiotics excreted by animal husbandry

adds up to thousands of tons per year, major concerns about their degradation in the

environment have risen during the last decades. One main problem with regard to the

excessive use of antibiotics in livestock production is the potential promotion of resistance

and the resulting disadvantages in the therapeutic use of antimicrobials. Since the beginning

of antibiotic therapy, more and more resistant bacterial strains have been isolated from

environmental sources showing one or multiple resistance. After administration, the


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xvii

medicines, their metabolites or degradation products reach the terrestrial and aquatic

environment indirectly by the application of manure or slurry to areas used agriculturally or

directly by pasture-reared animals excreting directly on the land.

After surface run-off, driftage or leaching in deeper layers of the earth, their fate and the

impact on environmental bacteria remains rather unknown. Especially against the background

of increasing antibiotic resistance, the scientific interest in antimicrobially active compounds

in the environment and the possible occurrence of resistant bacteria has increased

permanently. On the one side, scientific interest has focused on the behaviour of antibiotics

and their fate in the environment, on the other hand, their impact on environmental and other

bacteria has become an issue of research. With the advances of modern analytical methods,

studies using these new techniques provide accurate data on concentrations of antimicrobial

compounds and their residues in different organic matters. Some antibiotics seem to persist a

long time in the environment, especially in soil, while others degrade very fast. Not only are

the fate of these pharmaceuticals, but also their origin and their possible association with

resistant bacteria objects of scientific interest.

This chapter presents an overview of the present studies on the use of veterinary

antibiotics in agriculture, on the occurrence of antibiotic compounds and resistant bacteria in

soil and water and clearly demonstrates the need for further studies.

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