7

2. Review of Literature

Pharmaceutical products are subjected to microbiological contamination that can

represent a health to the consumer and cause product spoilage, an esthetic changes, and

possible loss of drug efficacy. Microbial contamination may originate from the raw

materials and excipients or may be introduced during manufacturing process such as from

contaminated equipment, operators, air, packaging materials, storage and use. The

microbial contamination of pharmaceuticals has been studied extensively during the past

30 years. Microbial contamination control in the pharmaceutical industry is a

multidisciplinary approach requiring the interaction of microbiology, engineering and

chemistry. Optimization of microbial contamination control requires the development and

implementation of systems leading to environmental fluctuations that will minimize or

eliminate microbial survival and growth. However, the presence of objectionable

microorganisms in non-sterile products or any type of microorganisms in sterile products

indicates lack of process control and system optimization. Identification of microbial

contaminants provides important information to track contamination sources, implement

proper corrective actions and to understand microbial community composition. The

detection of microbial contaminants has been traditionally performed using cultivation-

based methods. Traditional microbiological methods are labor intensive and time

consuming. However, new molecular methods are available that can rapidly detect

microorganisms in contaminated samples.

The present research work deals on the identification of contamination profile in

pharmaceutical environment; analyzing the contaminants by rapid molecular methods

and controlling the contamination by analyzing the efficacy of disinfectants. This part of

thesis brings out a brief narration of already available reports on the above aspects

appropriately.

8

2.1 Product recalls due to microbial contamination

The contaminating microorganisms may cause spoilage of the pharmaceutical

product with loss of its therapeutic properties and if they are pathogens in high number,

represents a serious health threat to consumers (patients), as products ingested or applied

to human skin (Denyer, 1990). On the basis of Food and Drug Administration (FDA) of

United States of America, recall data from 1998 to September 27, 2006, heterotrophic

microorganisms caused the majority of microbial contamination reported in non-sterile

pharmaceutical products. A summary of bacterial and fungal contaminants for non-

sterile pharmaceutical products in the United States is shown in Table 1 and 2. Some of

the products are liquids, tablets, capsules, oils, drops, creams and emulsions. The pH of

the recalled formulations ranged from acidic to alkaline. Evidently, microorganisms are

capable of contaminating a given pharmaceutical formulations regardless of water

content, pH, or manufacturing process (Sutton and Jimenez, 2012).

Of the 134 recalls reported by the FDA, 60% were associated with contamination

by Gram-negative bacteria, while Gram-positive bacteria were found in only 4% of

recalls. The numbers suggest that Gram negative bacterial contamination appeared to be

a more serious problem than those Gram-positive bacteria. The different types of

microbial species isolated from recall samples included were Pseudomonas spp.,

Burkholderia cepacia and Ralstona pickettii contamination, accounting 48 % (Table 1)

(Jimenez, 2004; Anonymous, 2007; Sutton and Jimenez, 2012). These contaminants are

commonly found in water samples. Water is known to be the most common raw material

in pharmaceutical manufacturing and also used for preparing disinfectant solutions,

cleaning equipments, floors, and walls. (Marino et al., 2000 and Kawai et al., 2002).

Contamination by yeast and mould was found to be the second cause for product

recall, although these contaminants not generally speciated (Table 2). Twenty-three

percent of non sterile products recalls were due to yeast and mould contamination. The

reported common mould contaminants are species of Aspergillus, and Penicillium

(Jimenez, 2004; Vijayakumar et al., 2012a). Recently, Khor et al. (2006) reported the

outbreak of Fusarium keratitis with substantial morbidity was associated with use of a

specific contact lens solution.

9

Table. 1 FDA Product recalls due to bacterial contamination in non-sterile and sterile

pharmaceutical products in the United States (from 1995 to 2006)

Product Reason / Contaminant

Acetaminophen Aerobic microorganism

Benzyl peroxide solution Burkholderia cepacia

Topical cream Pseudomonas putida

Triclosan lotion Pseudomonas aeruginosa

Acne cream B. cepacia

Albuterol sulfate inhalation solution B. cepacia

Albuterol sulfate syrup B. cepacia

Ursodiol capsules Potential microbial contamination

Vera Gel Enterobacter gergoviae

Nonalcoholic body spray B. cepacia

Triple S gentle wash P. aeruginosa

Sodium chloride cleanser P. aeruginosa

Albumin human 5% Enterobacter cloacae

Eye gel P. aeruginosa

Mouth rinse antiplaque alcohol-free B. cepacia

Medical food nutrition supplement P. aeruginosa

Dialysate concentrate Bacterial contamination

Tylenol gelcaps Aerobic microorganisms

Brand baby oil B. cepacia

Wet and wild liquid makeup P. aeruginosa

Topical product P. aeruginosa

F12 nutrient mixture Bacterial contamination

Gelusil liquid anti gas antacid Bacillus spp.

Hydrox alcohol-free mouthwash B. cepacia

Neoloid emulsfied castor oil Exceeds microbial limits

Mouth rinse alcohol-free B. cepacia

Fresh breath plus mouthwash P. aeruginosa

Fresh moment alcohol-free mouthwash B. cepacia

Children’s cologne P. aeruginosa

Mouth rinse antiplaque alcohol-free

Oral B B. cepacia

Aloe vera cream B. cepacia

Antacid–antigas liquid suspension Bacterial contamination

10

Sea therapy mineral gel P. aeruginosa, P. fluorescens

Shampoo exotic fruits Bacterial contamination

Mouth wash alcohol-free P. aeruginosa

Medical food nutrition supplement P. aeruginosa

Panama jack tanning lotion Bacterial contamination

Acne treatment cream B. cepacia

Clinical resource food supplement P. aeruginosa

Nystatin oral suspension Possible microbial contamination

Kenwood brand emulsified castor oil Exceeds microbial limits

Fluoride mouth rinse B. cepacia

Benzoyl peroxide wash Potential for microbial contamination

Shampoo (antidandruff) B. cepacia

Misoprostal tablets B. cepacia

Simethicone drops B. cepacia

Nutritional beverage powders May contain Salmonella spp.

Formance May contain Salmonella spp.

Cytotec tablets Pseudomonas spp.

Propac protein supplement Salmonella

Soylac infant formula May contain Salmonella

Ben-Agua wash Potential for contamination

Kayolin pectin suspension Microbial contamination

Antacid oral liquid suspension Bacterial contamination

Body wash and shampoo Klebsiella oxytoca

Eye shadow Pseudomonas stutzeri

Soy protein infant formula Klebsiella pneumoniae ,P. aeruginosa

Antacid–antigas oral Bacterial contamination

Aloe skin cream B. cepacia

Food industry sanitizing soap B. cepacia

Hand disinfectant and body lotion B. cepacia

Shampoo B. cepacia

Alcohol free mouthwash P. aeruginosa

Cough syrup Exceeds microbial limits

Disinfectant first aid treatment B. cepacia

Sunburn gel and spray B. cepacia

Antiplaque alcohol free mouth rinse B. cepacia

Infant formula Nonpathogenic spoilage microorganisms

Boric acid solution Exceeds microbial limits

11

Minocycline capsules Microbial contamination

Myla-care antacid antigas liquid Bacterial contamination

Sodium chloride Ralstonia pickettii

Benzalkonium chloride towelette B. cepacia

Calcitriol Bacillus cereus

Syrup Staphylococcus warneri

Haloperidol oral solution Microbial contamination

Hydrocortisone polistirex suspension Microbial contamination

Lidocaine HCl/epinephrine injection Microbial contamination

Colostrum cream P. putida

Eye and ear drops P. fluorescens

Ophthalmic solution B. cepacia

Antiseptic solution P. aeruginosa

Nystatin oral suspension Acinetobacter baumanii

Povidone–iodine solution P. putida,Salmonella spp., Aeromonas sobria

Bactroban ointment R. pickettii , P. fluorescens

Gel Microbial contamination

Simethicone solution Microbial contamination

Antacid liquid Bacillus licheniformis

Eye and nasal drops P. mendocina, Klebsiella pneumoniae

Mouthwash P. alcaligenes, P. baleurica

Nasal spray P.fluorescens

Antacid Liquids Enterobacter cloacae, Citrobacter freundii,

Klebsiella pneumonia, Flavimonas oryzihabitans,

Salmonella arizonae,

Medicated Hand wash P. spinosa

Antiseptic mouth wash Yeast and mould contamination

Nasal spray B. cepacia

Hand sanitizers Bacterial contamination

Nasal spray B.cepacia

Calcium carbonate, Simethicone solution S.aureus

Pharmaceutical topical creams Microbial contamination

Oral pharmaceuticals Microbial contamination

Antibacterial hand soap P. aeruginosa

Gel capsules P. aeruginosa

Oral pharmaceuticals P.aeruginosa, B.cepacia

Dimethicone solution B.cepacia

12

From a total of 197 recalls covering 1998 to 2006, the lack of sterility assurance

appeared to be the number one reason for product recalls (Jimenez, 2007). Over the last

8 years, 78% of sterile product recalls were due to lack of sterility assurance. Some of

the reasons given were package integrity deficiencies; media fill failures, improper

sterilization validation and numerous deficiencies during aseptic processing.

Gram negative bacteria were found in 6% of recalls, while Gram-positive bacteria

accounted for only 1%. The most abundant microbial species was B. cepacia with 2.5%

of recalls and Mycobacterium spp. accounted for 2%. Yeast and mould contaminations

were found to be responsible for 7% of recalls of sterile pharmaceuticals. It has been

reported that the presence of mould such as species of Penicillium and Aspergillus might

have indicated improper sanitization of surfaces and lack of controls in air circulations.

Any contamination in a sterile product is an unacceptable risk to patient safety, with non-

sterile products especially respiratory products; mould contamination will cause serious

issues to users (Vijayakumar et al., 2012a).

As a result of the increase in product recalls, the FDA has developed an upgrade

for a technical monograph on aseptic processing of sterile products (Akers, 2002). This

monograph further describes the critical control points during aseptic processing of

pharmaceutical products. Furthermore, the document provides guidance in many areas

where problems are persistent and redundant.

13

Table 2. Product recalls due to fungal contamination by FDA in the United States (from 1995

to 2009)

Product Isolate

Aminocarproic syrup Yeast (Candida parapsilosis)

Barium sulphate Mould

Dial brand dialyte concentrate Mould

Electrolyte solution Aspergillus niger

Dry skin cream Mould

Vinegar and water douche Mould

Preparation H ointment Mould

Penecare lotion Candida lipolytica

Aidex spray cleaner Mould

Astringent pad Mould

Oral suspension Yeast

Vitamin E lanolin lotion Mould

Hand and body lotion with lanolin Mould

Sodium fluoride oral mouth Mould

Bicarbonate suspension Mould

Ampicillin suspension Mould

Progesterone cream Mould

Baclofen & Methylprednisolone injection Penicillium spp.

Glycyrrhizinic acid injection Mould

Human tissue processed by Cryolife, Inc. Mould

Orasept antiseptic mouthwash and gargle Yeast and mould

Starbrite brand Black Magic Color Tattoo Ink Acremonium & P.aeruginosa

Medline Baby lotions and oils B. cepacia & Fungus

Bausch & Lomb contact lens cleaning solution Fusarium

Lubriderm Moisture Mitts, skin moisturising

lotion

A. fumigatus, A. versicolor

Penicillium spp.

Allopurinol – tablets (brand name: Purinol) Rhizopus microsporus

Medical devices Apergillus spp., Penicillium spp.

14

2.2 Sources of microbial contamination

One of the most important areas in pharmaceutical process control is the

development of systems to control the number, survival, and proliferation of

microorganisms during manufacturing of non-sterile and sterile pharmaceutical products.

In relation to this general profile, commonly considered four main sources of microbial

contaminations are clean room air, personnel, surfaces and water.

Sandle (2011a) reported that studying the range, types and patterns of

microorganisms found in clean rooms can provide essential information for

microbiologists and quality control personnel in understanding the clean room

environments and for assisting with contamination control. In an earlier study, Nagarkar

et al., (2001) pointed out that maintaining the integrity of a pharmaceutical production

environment of cleanroom is a constant battle. To decide which method or combination

of methods to be employed in disinfecting aseptic workshop, there is need to understand

the kind of bacteria and fungi that are the prime sources of contamination. Therefore,

knowledge of the microbial diversity of cleanrooms, as well as any extreme

characteristics these microbes might possess, is essential to the development of

disinfection technologies.

Most common microorganisms in cleanrooms are Gram-positive bacteria. These

microorganisms often have a close phylogenetic affiliation as indicated by comparative

analysis of partial 16S rDNA studies, such as between the Micrococci and Staphylococci

(Clarridge, 2004). In addition, there are, in fewer numbers, certain fungi associated with

cleanrooms. Cleanroom microflora is predominantly of Gram-positive bacteria (Wu and

Liu, 2007). Several authors reported the common species included were species of

Micrococcus, Staphylococcus Corynebacterium, Bacillus, Aspergillus and Penicillium

(Hyde, 1998; Johnson, 2003 and Wu and Liu, 2007).

With the genera Staphylococcus and Micrococcus, many of the species are

indigenous to humans (Heikens et al., 2005). Although Gram-positive microorganisms

are ubiquitous in cleanrooms and make up the overwhelming majority of isolates, there is

15

little published work relating to the expected proportion of microorganisms.

Utescher et al. (2007) reported that the characteristic microbial population identified in

these monitored areas composed of the bacteria such as Staphylococcus spp.,

Micrococcus spp., and Bacillus spp. Wu et al. (2007) reported that the predominant

contaminant bacteria were a group of Gram positive bacteria either spore-forming

Bacillus or non-sporulating Staphylococcus and Microbacterium. Only few studies

reported worldwide, because pharmaceutical companies are not willing to share their

confidential reports.

Very few articles are available regarding mould contamination incidences or

identification issues from pharmaceutical clean room environments. Therefore, it is

difficult to review the aerobiocontamination data in region wise. Vijayakumar et al.

(2012a) reported that species of Aspergillus, Cladosporium, Penicillium, Alternaria,

Curvularia and Fusarium were the most predominant fungal isolates from

pharmaceutical processing environments. Utescher et al. (2007) reported that species of

Cladosporium, Aspergillus, Scopulariopsis, Fusarium, Alternaria, and Mycelia sterillia

were found in low frequencies and predominantly in Grade D/ISO Class 9 environments.

However, various researchers reported the profile of fungal contaminations from

non clean rooms such as air handling units, hospitals and outdoor environments

(Ortiz et al., 2009; Qudiesat et al., 2009; Sautour et al., 2009; Kim et al., 2010).

The second critical area is the personnel who are the major sources of

contamination during manufacturing (Hyde, 1998). There are some genera of bacteria

which are generally represented (Grice et al., 2008 and 2009). When research of the

bacteria biota of human skin is compared with published work of cleanroom

microorganisms, there is an association between the microorganisms commonly found in

cleanrooms and those which are transient to (short-term or long term-residents on) human

skin (Owers et al., 2004; Moissl et al., 2007). Some of the species living in the human

skin are Staphylococcus epidermidis, S. capitis, S.hominis, Propionibacterium spp.,

Propionibacterium acnes, Micrococcus spp., etc. The normal flora for the human oral

cavity is comprised of Streptococcus salivarius, S. mutans etc. Moulds can also be

16

possible contaminants. Common moulds from human flora are Trichophyton spp.,

Epidermophyton spp., Microsporon spp., etc. To protect critical areas from human

microbial flora, personnel wear gowns, hair covers, hoods, shoe covers, laboratory coats,

face masks, gloves, boots, etc. In addition other types of microorganisms present in

cleanrooms, such as Bacillus spp. (La Duc et al., 2009), are those present in soil. Such

microorganisms may be transferred into the cleanrooms via personnel, dust, and material

transfer (Halls, 2004; Haberer, 2008).

The third area of concern is the equipment and building areas. Unless equipment

is cleaned and sanitized, there is always the risk of microbial contamination. However,

cleaning and sanitization of the equipment must provide a hostile environment for

microorganisms to survive and grow. Bacteria such as Pseudomonas spp., S. epidermidis,

Bacillus spp., etc. are commonly found in equipment. Moulds are commonly found

in walls and ceilings. Continuous sanitization and disinfection of floors, drains walls and

ceilings are advised to avoid the microbial colonization of these areas. Some of the

mould species are Aspergillus spp., Penicillium spp., and Aureobasidium spp.

(Underwood, 1998).

The great majority of the microbial contamination for nonsterile products has

been reported to be due to the presence of microorganisms in raw materials or water or

from poor practices during product manufacturing (Baird, 1998). Manufacturing under

nonsterile conditions requires operators to follow specific GMP practices such as raw

material testing, equipment sanitization, and wearing of gloves, masks and laboratory

uniforms. Different types of bacteria commonly found in pharmaceutical raw materials

are Lactobacillus spp., Pseudomonas spp., Bacillus spp., Escherichia spp., Streptoccocus

spp., Clostridium spp., Agrobacterium spp., etc. and moulds such as Cladosporium spp.

and Fusarium spp. (Vijayakumar et al., 2012a).

Various authors reported that water is known to be the most common raw material

in pharmaceutical manufacturing. Water is also used to rinse and clean equipment,

floors, and walls (Marino et al., 2000 and Kawai et al., 2002). In lower-grade

cleanrooms, where there is a water source, some microorganisms associated with water

systems will be detected (Jimenez, 2004).

17

2.3 Diagnosis of microbial contamination and Identification of contaminants

Diagnosis of contamination helps to establish a “norm” and provide a measure for

trending purposes such as noting the frequency of occurrence of isolates by genera or

species over time and across cleanrooms or locations within cleanrooms (Jimenez, 2004).

Furthermore, the need for microbial identification is detailed in a number of

pharmacopoeial chapters in both the Indian Pharmacopoeia (IP) and in the United States

Pharmacopoeia (USP). USP Chapter 1116 addresses establishing the normal microbial

flora and using microbial identification to assess the effectiveness of the cleaning and

sanitization program and to investigate the source of microbial contamination, especially

when environmental monitoring action levels are exceeded (Anonymous, 2007 and

2010).

Testing must be performed to determine the quality of these materials. The

absence of E. coli, S. aureus, P. aeruginosa, and S. typhimurium is required before raw

materials can be used in pharmaceutical products. Standard methods are used in clinical,

environmental, pharmaceutical, and food microbiology to diagnose microbial

pathogenesis and contamination (MacFaddin, 1985; Jimenez, 2004 and Koneman and

Elmer, 1997).

The presence of objectionable microorganisms in cosmetic and pharmaceutical

products represents a serious health threat to consumer’s worldwide (Underwood, 1998;

Kamil, O.H. 2011). Furthermore, microbial growth has a negative impact on product

integrity (Sutton, 1997). Over the last 30 years, implementation of GMP has been the

foundation for improving industrial quality control analysis. As part of GMP, the USP

and IP Microbial Limits Test provide methods for the determination of total microbial

counts for bacteria, yeast, and mould (Anonymous, 2007; 2008 and 2010). In addition to

the microbial content, microbiological analysis generally determines the safety of a

product through the absence of indicator microorganisms, which can be considered a

hazard to consumers and indicative of contamination. For this purpose, the USP and IP

specify 4 bacterial indicators: Salmonella spp., S.aureus, P. aeruginosa, and E.coli. The

18

European Pharmacopeia (EP) specifies these same 4 bacterial indicators, including an

additional requirement for ascertaining the different levels of Enterobacteria

(Anonymous, 2004). On the basis of published scientific studies, the Enterobacteriaceae,

Pseudomonas spp., B. cepacia and mould have been found to be the most common

microbial contaminants around the world (Abdelaziz et al., 1989; Oie and Kamiya, 1996;

Zani et al., 1997 and Jimenez, 2007). The detection of microbial contaminants has been

traditionally performed using cultivation-based methods (Baird, 1998).

According to the EP, IP and USP pharmacopoeias, for a non-sterile

pharmaceutical product, microbial limit testing is performed in a stepwise manner; first,

the sample is tested to determine the number of microorganisms (Anonymous, 2004;

2006; 2007 and 2010). This will indicate how many bacteria, yeast, and moulds are

present in a sample. Second, for qualitative analysis, the sample is incubated in broth for

at least 24 hours to enhance the isolation of some pathogenic microorganisms. The reason

for incubating the samples for at least 24 hours is due to the fact that pathogenic

microorganisms are present in lower numbers than nonpathogenic microbes. An

enrichment step and growth on selective medium will enhance the isolation of pathogenic

microorganisms such as Salmonella spp. and E. coli. Before sample testing is performed,

the methods must be shown to be capable of detecting and isolating bacteria, yeast and

mould (Anonymous, 2007).

Standard methods are based upon the enrichment, incubation and isolation of

microorganisms from pharmaceutical samples. Because of the long incubation times,

continuous manipulation and time-consuming procedures, results are normally obtained

within 6 to 8 days. Various researchers reported that standard methods underestimate the

microbial communities present in pharmaceutical environments (Nagarkar et al., 2001;

Kawai et al., 2002 and Venkateswaran et al., 2003). This has been demonstrated in

samples of water, contact plates and air samples from different pharmaceutical

manufacturing facilities and clean room environments.

During the last five years, several peer review studies have been published on the

research, development, validation and application of rapid methods to pharmaceutical

microbiology (Samadi et al., 2007; Karanam et al., 2008 and Ragheb et al., 2012). ATP

bioluminescence, direct viable counts, deoxyribonucleic acid (DNA) and PCR

19

technology have demonstrated that a non culturable portion of the microbial community

in pharmaceutical environments is viable and undetectable by compendial methods.

Therefore, these new technologies provide a higher resolution and discrimination

between microbial species.

The new molecular methods are available that can rapidly detect microorganisms

in contaminated samples. ATP bioluminescence and PCR based assays have been

developed and validated for rapid microbiological screening of clinical and food samples

(Hill, 1996, Ieven and Goosens, 1997).

Hugo (1998) reported that ATP is the most important high-energy phosphate

compound present in a microbial cell. ATP carries an important function in the microbial

cell by providing the energy source to drive microbial viability and growth. ATP

bioluminescence technology is based upon the reaction of the enzyme complex

luciferase–luciferin, in the presence of oxygen and magnesium, with ATP released from

microbial cells resulting in the production of light. Several studies have demonstrated the

applicability of ATP bioluminescence to pharmaceutical quality control and it has been

previously used as an indicator of microbial viability and biomass in environmental

studies (Underwood, 1998). Other studies have reported on the use of ATP

bioluminescence assays for determination of the microbial content of different raw

materials (De La Rosa, 1995).

Ignar et al. (1998) reported the detection of bacteria, yeast, and mould

contamination in pharmaceutical products within 24 to27 hours using the Celsis ATP

bioluminescence system (Celsis, Inc., Evanston, IL) and specific enrichment broths. ATP

bioluminescence assay provided a 24 hours count of bacteria present in water samples

from a reverse osmosis/ ultrafiltration water system, hot water circulating system and

cold tap water (Scalici et al., 1998). ATP bioluminescence has also been used for rapid

sterility testing of pharmaceutical suspensions and microbial content analysis of finished

products (Ignar et al., 1998). However, ATP bioluminescence provides an indication of

the total microbial biomass in a product which might be including microbial species

whose presence can be acceptable for non-sterile pharmaceuticals. Marino et al. (2000)

reported that ATP bioluminescence assays have been used for rapid monitoring of quality

in pharmaceutical water systems. Compared with standard methods, which require more

20

than one growth media, the ATP bioluminescence assays were capable of detecting all

microorganisms using a single growth medium in a shorter period of time. Drawback of

this method is ATP extraction and detection from environmental samples is a labor-

intensive and time-consuming procedure.

2.3.1 Molecular diagnosis of microbial contamination - Nucleic acid amplification

techniques (PCR Technology)

Molecular diagnostic tests based on nucleic acid amplification technologies

(NAAT) have become widely established in clinical microbiology laboratories in recent

years, as well as in quality control (QC) laboratories for food testing and have been

offered lately to the QC laboratories in the biopharmaceutical sector (Denoya, 2009).

NAAT rely on the reiteration of the process of DNA polymerization, leading to

exponential increase of a specific fragment of the nucleic acid, i.e the use of the PCR.

Hill (1996), and Ieven and Goosens (1997) have reported that PCR based assays are used

routinely in the food industry and clinical laboratories to detect and identify pathogenic

bacteria, yeast and mould.

The first PCR application to cosmetics and pharmaceutical quality control has

been reported by Jimenez et al. (1998). They used the BAXTM system (Dupont Qualicon,

Wilmington, DE), a PCR-based assay, S. typhimurium was detected in all 25 samples of

raw materials and finished products after a 24 hours enrichment. This represented a faster

turnover time than the standard 5 to 6 day detection time. PCR based assays were also

developed and validated to detect all other 3 USP bacterial indicators, E. coli, P.

aeruginosa, and S. aureus in samples of 24 various pharmaceutical raw materials and

finished products (Jimenez et al., 1999a and 2000b). They detected in artificially

contaminated samples of finished products and raw materials within 27 to 30 hours.

Jimenez and Smalls (2000b) have reported the simplified method with Ready-To-

Go PCR beads to detect of B. cepacia in artificially contaminated pharmaceutical

samples. Although the USP and EP required the absence of the 4 bacterial indicators,

21

published scientific studies demonstrated that B. cepacia is one of the more frequently

isolated bacterial contaminants in cosmetic and pharmaceutical samples in the United

States (Orth, 1996; Palmieri et al., 1988). Furthermore, B. cepacia is found to be a major

contaminant in samples of finished products from around the world (Zani et al., 1997;

Underwood, 1998). Standard methods for isolation and identification of B. cepacia

require 5 to 6 days to be completed. However, PCR detection of samples contaminated

with B. cepacia was completed within 27 hours using the Ready-To-Go PCR beads.

Sample contamination by moulds is a major concern due to the widespread

distribution of spores, strain pathogenicity and their effect on product integrity. However,

detection of mould contamination in raw materials and finished products require 6 to 8

days to be completed (Anonymous, 2007). Jimenez et al. (1999b) developed PCR assay

to detect mould contamination (A. niger)in pharmaceutical samples. In that study test was

completed within 27 hours while the standard methods required 6–8 days. Yeast

contamination, e.g., Candida albicans, was also detected in all the contaminated samples

using a PCR-based assay (Jimenez et al., 1998).

Jimenez (2000a) used a PCR-based amplification of conserved ribosomal

bacterial sequences (1.5 kb); it is possible to confirm sterility. All tested samples that

were positive by the PCR reaction were also positive by conventional methods. PCR-

based assays reduced turn over time from 72 to 24 hours.

Simultaneous PCR detection of bacteria and mould from contaminated

cosmetic/pharmaceutical samples was performed using the Stratagene Robocycler 96-

Gradient. This new thermocycler allowed the simultaneous amplification of genetic

sequences using DNA primers with different annealing temperatures. Low levels of

microbial contamination ranging from 1 to 7 CFU were detected (Jimenez, 2001b).

Various finished products and raw materials have been assayed by PCR in cosmetic and

pharmaceutical laboratories by Jimenez (2001a).Samadi et al. (2007) reported PCR based

detection of low levels of S. aureus contamination in topical lotion pharmaceutical

preparations.

22

There were no reports available for ophthalmic products, chemotherapeutic and

gastrointestinal drugs. However, Vijayakumar et al. (2011a) developed PCR to detect the

low levels of P. aeruginosa contamination in ophthalmic viscosurgical devices by using

universal and specific primers (oprL) to P. aeruoginosa as one of the objectionable

microorganisms in pharmaceutical products. The feasibility of PCR-based detection of

different types of microorganisms is being demonstrated based on recent reports in the

scientific literatures listed in Table 3.

Table. 3 Pharmaceutical products analyzed by PCR methods by various authors

Test products PCR for Pre-enrichment

step included PCR

Inhibition Reference

Fluoride dentifrice, Medicated dentifrice, Sleeping tablet, Antiflatulent liquid, Carboxymethylcellulose, Simethicone and Lactose

B. cepacia

Bacteria and Mould

Yes No Jimenez and

Smalls (2000)

Vee gum, Carboxymethylcellulose Silica, Starch, Simethicone emulsion, Antiflatulent liquid

Salmonella typhimurium

Yes No Jimenez et al.,

(2001)

Topical lotion S. aureus Yes No Samadi et al.,

(2007)

Lactose, Nicotinamide, Sodium starch glycollate, Ranitidine HCL, Mannitol, Ibuprofen suspension

S. aureus, Salmonella spp.

E. coli P. aeruginosa

Yes No Karanam et al.,

(2008)

Expectorant syrup

S. aureus, Salmonella spp.

E. coli P. aeruginosa

Yes No Farajnia et al.,

(2009)

Hydroxypropyl mehtyl cellulose and Sodium Hyaluronate

P. aeruginosa No No Vijayakumar et

al., (2011a)

23

2.3.2 Multiplex PCR

Multiplex PCR assays have been developed and validated for environmental, food

and clinical analyses (Knabbel and Crawford, 1995; Mahony et al., 1995and Brasher et

al., 1998). So there is need to develop a multiplex assay to amplify all relevant

pharmaceutical and cosmetic microbial contaminants in one assay.

Jimenez et al. (2000b) developed PCR analysis for detecting low levels of

bacteria and mould contamination in pharmaceutical samples using Ready-To-Go PCR.

In this method samples were artificially contaminated with less than 10 CFU of E.coli,

S.aureus, P.aeruginosa and A.niger. Standard methods required 6 – 8 days while PCR

method detects the contaminants within 27 hours.

Recently Farajnia et al. (2009) carried out a study on simultaneous detection and

identification of four indicator pathogenic bacteria in a single PCR reaction in

expectorant syrup. They used specific primers for four USP indicator bacteria and their

detection time was 27 hours. In India, a study from Hyderabad, Karanam et al. (2008)

developed multiplex PCR for detection of indicator pathogens from various raw materials

and finished products. In their study, the detection limits for artificial contaminants was

1 CFU/g, whereas in the case of conventional method, the detection limit was > 2 CFU/g.

Similarly, when tested with possibly contaminated samples, 35% was detected for E.coli,

Salmonella spp., S. aureus and P. aeruginosa with multiplex PCR, while only 21% was

detected with standard conventional microbial methods.

Furthermore, the recent advances in DNA microchip technology can lead to

specific assays for quality control purposes. DNA microchips have already been used for

detecting multiple microbial populations in environmental and clinical samples (Guschin

et al., 1997). The same technology might be capable of detecting microbial contaminants

in pharmaceutical samples.

24

2.4 Contamination control

Antiseptics and disinfectants are used extensively in hospitals and other health

care centers to control the growth of microbes on both living tissues and inanimate

objects. They are essential parts of infection control practices and aid in the prevention of

nosocomial infections (Larson, 1991). However, a common problem is the selection of

appropriate disinfectants and antiseptics because different pathogens vary in their

response to different antiseptics or disinfectants (Russel, 1995 and 1996 a&b). Recently,

Sandle (2004) has reported that one of the more difficult tasks facing pharmaceutical

organizations is with the selection of disinfectants, particularly in ensuring that the

disinfectants selected are appropriate and that the effectiveness of the disinfectants are

periodically assessed. In a similar way to pharmaceutical industries, cleaning and

disinfection measures are important and decisive process steps for fulfilling the quality

requirements of the medicinal product. In order to decide which method or combinations

of methods are to be employed in disinfecting aseptic processing areas, it is important to

understand the types of micro-organisms that are the prime sources of contamination

(Nagarkar, 2001; Anonymous, 2007).

According to USP chapter 1072 and the European Commission’s good

manufacturing practice (EUGMP) guidelines, monitoring of environmental isolates and

checking their susceptibility pattern to disinfectants is very important for clean room

disinfection programs (Anonymous, 2007 and 2008). Antimicrobial susceptibility tests

are performed on bacterial and fungal pathogens in clinical microbiology setups,

especially if they belong to a species exhibiting resistance to commonly used disinfectant

agents. The susceptibility testing is also important in resistance surveillance,

epidemiological studies and in comparison of the in vitro activity of new and existing

agents. Dilution methods are used to establish the minimum inhibitory concentrations

(MICs) of antimicrobial agents; these are the reference methods for antimicrobial

susceptibility testing and are mainly used to establish the activity of a new antifungal

agent, to confirm the susceptibility of microorganisms to the antifungal agent that give

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equivocal results in routine tests, and to determine the susceptibility on fungi where

routine tests may be unreliable.

Unlike most antibiotics, few biocides exert their action upon one specific target

within the microbial cell. Most agents are capable of acting at several sites within the cell

and the interaction responsible for cell death is not always clearly established (Maris,

1995). Recent scientific evidence suggests that during the last decade, antibiotic

resistance by various mechanisms has increased worldwide in bacterial pathogens leading

to treatment failures in human and animal infections. However, the resistance against

different types of biocides (including disinfectants, antiseptics, preservatives and

sterilants) has been studied and characterized. Only limited sound scientific evidences to

correctly weigh the risks of antibiotic resistance induced by resistance to biocides is

available and some controversies remain (Russell, 1995 and 1996b; Mc Donnell and

Russell, 1999 and Joynson et al., 2002).

2.4.1 Antibacterial efficacy of common biocides

The concentration of a biocide has been deemed to be the most important factor

that affects its efficacy (Russell and McDonnell, 2000). Concentration is also central to

the definition of bacterial resistance in practice. Therefore, the measurement of bacterial

lethality rather than the measurement of bacterial growth inhibition is paramount. Many

reports on emerging bacterial resistance to biocides are based on the determination of

MICs. Using MICs to measure bacterial resistance is arguable since much higher

concentrations of biocides are used in practice and, therefore, failing to achieve a

reduction of bacterial numbers (i.e. lethality) because of elevated MICs is unlikely

(Russell and McDonnell, 2000). Indeed, some studies have shown that bacterial strains

showing a significant increase in MICs to some biocides were nevertheless susceptible to

higher (in use) concentrations of the same biocides (Thomas et al., 2005; Lear et al.,

2006).

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MIC determinations have been used in many studies as an indicator of bacterial

sensitivity change to a biocide. Bacteria showing an increased low-level of

resistance/tolerance to a biocide might be selected by a low concentration of a biocide.

Their level of resistance can increase through selection, for example by repeated

exposure to a low concentration of a biocide or to increasing concentrations of a biocide

(Russell and Mcdonnell, 2000, Walsh and Fanning, 2008). The determination of

bacterial growth kinetics in the presence of a low concentration of a biocide can also

provide indications of a change in bacterial phenotype (Maillard, 2007).

Chlorhexidine is probably the most widely used biocide in antiseptic products, in

particular in hand washing and oral products but also as a disinfectant and preservative.

This is due in particular to its broad-spectrum efficacy, substantivity for the skin and low

irritation. Of note, irritability has been described and in many cases may be product

specific (Rosenberg 1976; Gardner and Gray, 1991). Chlorhexidine is a bactericidal

agent. Its interaction and uptake by bacteria were studied initially by Hugo et al.(1966)

who found that the uptake of chlorhexidine by E. coli and S. aureus was very rapid and

depended on the chlorhexidine concentration and pH.

Hiom et al. (1993 and 1995) have reported the effects of chlorhexidine on yeast

cells and the results are probably similar to those previously described for bacteria.

Increasing concentrations of chlorhexidine (up to 25 mg/ml) induce progressive lysis of

Saccharomyces cerevisiae protoplasts, but higher biguanide concentrations result in

reduced lysis. Russell (1990 and 1995) reported that chlorhexidine has little effect on the

germination of bacterial spores but inhibits outgrowth. The reason for its lack of effect on

the former process but its significant activity against the latter is unclear. It could,

however, be reflected in the relative uptake of chlorhexidine, since germinating cells take

up much less of the bisbiguanide than do outgrowing forms (Shaker, 1988).

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Quarternary ammomium compounds (QACs) have been used for a variety of

clinical purposes and excellent for hard-surface cleaning and deodorization. It has been

known for many years that QACs are membrane active agents (Hugo and Frier, 1969).

QACs are also believed to damage the outer membrane of Gram-negative bacteria,

thereby promoting their own uptake. McDonnel and Russell (1999) reported that Gram-

negative bacteria tend to be more resistant than Gram-positive organisms, such as

staphylococci and also reported MIC of selected biocides.

2.4.2 Antifungal efficacy of common biocides

The structure and function of the fungal wall have been studied in depth (Brul and

Klis, 1999). The fungal cell wall is a dynamic structure and can adapt to different

physiological states (e.g. sporulation) or morphological changes, e.g. hyphal growth for

yeast such as C. albicans (Klis, 1994; Molina et al., 2000). The fungal cell wall

demonstrates mechanical strength and a close relationship exists between wall

composition and taxonomic classification (Russell 1999). Other components are

associated with the fungal cell wall, of which melanins and sporopollenin might be

involved with cellular resistance to physical and chemical agents (Russell and Furr 1996).

The activity of biocides against fungal microorganisms is not as well documented

as their activity against bacteria. In general, fungi are more resistant to biocides than non-

sporulating bacteria. Only few studies are available for lethal concentrations of

antiseptics and disinfectants towards yeasts and moulds (Wallhausser, 1984; Vijayakumar

et al., 2011b). Vijayakumar et al. (2012 b) studied the MIC of biocides to clean room

fungal isolates and they reported that MICs of chlorhexidine, benzalkonium chloride and

cetrimide were in the range of 8 –16 µg/mL against hyaline fungi while the MIC range of

biguanides and QACs against dematiaceous fungi was ranging from 8 to 16 µg/mL.