Isolation and Identification of Multi-Drug Resistant Strains of Non-Lactose Fermenting Bacteria from Clinical Isolates

Purpose: We studied the drug resistance of different microbes from clinical isolates. The morphological characteristics of bacteria were observed through culture characteristics and by carrying out gram staining techniques while the biochemical characteristics of bacteria were carried out by biochemical test. Methods: A total of 324 samples were collected from suspected patients visiting different hospitals at district Peshawar. For morphological identification, samples of clinical isolates were analyzed by blood agar, MacConkey agar and Eosine Methylene Blue, identified by gram staining and characterized by different biochemical tests. Antibiotic Sensitivity test by Modified Kirby-Bauer Disc diffusion method was used to test the in-vitro susceptibility of the identified isolates to different antibiotics such as Ceftazidime, Ceftazidime, Ceftriaxone, Cefepime and Imipenem. Results: These resistant non-lactose fermenting gram negative bacteria were isolated from samples of pus/wound (33.30%, n = 108/324), blood (33.30%, n = 108/324), urine (23.30%, n = 75/324) and from ascetic/pleural fluids (10.20%, n = 33/324). The study revealed that the percentage of non-fermenting bacterial infection was higher in females (53%) as compared to males (47%) along with higher infection observed in the age group of 11 30 years. Pseudomonas aeroginosa showed high resistance against Cefepime (88.80%), followed by Cefoperazone (55.50%), Ceftazidime (48.10%), Ceftriaxone (33.30%). Imipenem was active with low resistance (7.40%). More resistance was seen in Morganella morganii against Imipenem (66.70%) followed by CefopeCorresponding author.


Introduction
Developing resistance to antibiotics is natural to microbes, which cannot primarily be ceased of as constantly evolving nature of microbes to chemicals around them.The phenomenon is very important regarding its practical and economic implications.It is because of this resistance people cannot be affectively treated and remain ill for a longer period of time.The development of tolerance of microbes to more than one drug is multi drug resistance (MDR) [1].In the last few decades, antibiotic resistance is becoming a major problem across the globe [2].
Factors leading to antibiotic resistance include a widespread and aggressive use of broad-spectrum antibiotics, wrong investigated and diagnosis, using drugs without proper prescription by physicians and doctors, further including misuse of drugs by patients.With the resistant strains of bacteria the treatments of common infections become difficult or impossible.Non-fermenters are gram-negative bacteria that cannot ferment sugars to produced energy for cell physiology.Gram negative non-fermenting bacteria (NFGNB) were isolated from different clinical samples.Because of extreme multidrug resistance problems, species of this group offer a serious challenge for healthcare management [3].As mostly, non-fermenting (gram-negative) bacteria are niche pathogens that cause infections in critically ill or immune-compromised patients.As they are primarily healthcare associated pathogens, they rarely cause infection in healthy individuals [4].
There are different mechanisms for resistance in non-fermenting gram-negative bacteria, including (i) production of enzymes (ii) enzymatic inactivation of antimicrobial agents (iii) specific targeted enzyme that is inhibited by antimicrobial agents (iv) alterations in target sites, (v) production of efflux pumps (vi) loss of outer membrane proteins or porins (vii) reduced uptake of the antimicrobial agent.That is because of these different resistance mechanisms that the therapeutic options are severely limited to treat infections caused by them [3] [4].Non-fermenters include many species belonging to several genera.Previous studies suggest four species rarely found in hospitals, significant problems in hospital practice, including Pseudomonas aeroginosa, Morganella morganii, Proteus mirabilis and Salmonella typhi [5].The emergence of multidrug resistance P. aeruginosa has emerged as a severe health problem [6], which is of low permeability of the cell wall, mutation in chromosomal genes regulating resistance genes and also acquiring resistance genes from other organisms via plasmids, transposons and bacteriophages [7] [8].As the infection caused by multiple antibiotics resistant P. aeroginosa may result in worse clinical outcomes, this bacterium has gained particular importance [9].Multi-antibiotic resistant of M. morganii strain is due to change in outer membrane permeability and mutation of the major porin or by a change in the number of porins in the outer membrane [10].Increasing resistance to β-Lactam antibiotics in P. mirabillis, is mediated by the production of acquired β-lactamases.Plasmid-mediated ESBLs, including TEMtype derivatives active against expanded spectrum cephalosporin are also spreading in P. mirabilis [11].
In Pakistan the first case of multidrug resistance of S. typhi was reported in 1987 [12].Since 1991, the cases of infections caused by Salmonella were increasingly resistant to extended-spectrum cephalosporins and fluoroquinolones [13].Salmonella typhi resistance to Fluoroquinolone is associated with point mutations occuring mostly within a domain of gyrA.Cephalosporins resistance is usually mediated by extended spectrum β-lactamases derived from TEM-and SHV-type enzymes [14].The present study aimed to assess the multi-drug resis-tance of non-lactose fermenting bacteria in a low economic country like Pakistan, where people are not well aware of health issues.Our investigation will be a real contribution to the society and human health for further proper medication and human ease.

Sample Collection
A total 350 clinical samples were collected from suspected patients in different hospitals of Peshawar, KP Pakistan.Samples were collected from urine, pus/wound, blood, ascetic/plural fluids and were analyzed for colonial morphology and routine biochemical identification.The isolation of clinical samples was carried out according to standard protocol [15].The collected samples from urine, pus/wound, blood and ascetic/plural fluids were spread on blood, MacConkey and Eosine Methyline Blue (EMB) agar plates and incubated at 37˚C for 24 -48 hours.Gram staining was carried out as early described [16] to identify the NFGNB bacteria.

Biochemical Characterizations
Biochemical characterizations were performed through biochemical tests of clinical isolates.The protocol for clinical sample's identification was according to Cheesbrough et al. [15].Indole, Methyl red, Citrate utilization, Triple sugar iron, Oxidase, Urease and Nitrate tests were also carried out.

Antibiotic Sensitivity Test
The Kirby-Bauer Disc Diffusion Method was used to test the in vitro susceptibility of the identified isolates to Ceftazidime (30 µg), Cefoperazone (75 µg), Ceftriaxone (30 µg), Cefepime (30 µg), Imipenem (10 µg).Pseudomonas aeroginosa colonies were picked up from the culture plate with the help of a sterile platinum wire loop and emulsified in 4 ml of sterile peptone water to match with 0.5 McFarland turbidity standards (1.5 × 10 8 cfu/ml).The surface of Mueller Hinton Agar (Oxoid, Basingstoke, UK) in a Petri dish was inoculated evenly through a sterile swab and for 10 minutes was allowed the agar to dry.A multichannel disc dispenser (Oxoid, Basingstoke, UK) was used to deposit the antibiotics discs onto the surface of the inoculated medium.The plate was then incubated at 37˚C for 24 hours.With measuring scale the diameters of zone of inhibition were measured in millimeters after 24 hours of period of incubation [17] [18].The above procedure was repeated thrice for each P. mirabillis, M. morganii and for S. typhi isolates.

Results
A total of 324 multiple-drug resistant gram negative non-fermenters were isolated from 350 clinical samples processed at Microbiology Laboratory of Services Hospital Lahore.These bacterial strains were then identified on the basis of their cultural, morphological and biochemical characteristics (Table 1).
The occurrence of isolates was higher in females (53%, n = 171 in 324) as compared to males (47%, n = 315 in 324), while highest frequency of MDR-NFGNB was observed among young patients of age 11 -30 (n = 171).The distribution of isolates among different age group showed that overall infection rate was higher in young individuals (11 -30 years) but P. aeroginosa infection was higher in old age (61 -74) (Table 1).
The Distribution of MDR-NFGNB P. aeroginosa, M. morganii, P. mirabillis and S. typhi in different clinical specimens (urine, pus/wound, blood, ascetic/pleural fluids) are shown in (Table 2).The results were also presented on simple cylindrical bar graph in Figure 1.

Discussion
Multi-drug resistance in bacteria is of very much concern to clinicians.Not only these organisms are very resistant but they are also rapidly spreading [19].In clinical practices the antibiotic resistant pathogens causes financial burden, treatment failure and can spread to other patients [20].In the last few decades, due to the widespread use of antibiotics, non-fermentative gram negative bacilli have emerged important health care-associated pathogens.Recent studies conducted on important areas like identification of non-fermentative gram negative bacilli, and monitoring their susceptibility patterns, which is important for the appropriate management of the infections caused by them, and to make clear the fact that it is important to establish the clinical relevance of the isolated non fermentative gram negative bacilli, before they are considered as pathogens.This would prevent unnecessary usage of antibiotics and the rise of drug-resistant strains [21].There is limited data on the prevalence and resistance pattern of NFGNB.The current study was intended to address these issues.
In this study MDR-NFGNB was isolated from patients of different age groups (11 -74).The highest number of MDR-NFGNB was isolated from age group of 11 -30 years, followed by age group of 31 -60 and 61 -74 years.People were prone to infection at the age of 11 -30 years because at this age the people are more active, have more social contacts, more journey from one place to another so have more chances of getting infection.Chances of getting infection do not depend upon time but depend on number of exposure to the injurious bacteria, viruses and toxins.At old age the people are restricted from social contact so have less chances of developing infection.
In the present study, Gender wise distribution of MDR-NFGNB was isolated both from males and females and highest numbers are from females.This could be due to the social activity of females in their life in developing countries like Pakistan.Where females are much ignored as compared to male so their food cleanliness is not good as males, as a result their immune system is weak.Furthermore, females give birth child's so admitted frequently to hospitals and thus has more chances of infection.
In-vitro MDR-Patteren in P. aeroginosa in present study to Imipenem was 7.40% which is consistence with the results of Romao et al. [28] who found 11% resistance against Imipenem, Tripathi et al. [29] found 10.80% resistance and Manian et al. [30] found 8% resistance against Imipenem in P. aeroginosa.Pseudomonas aeriginosa showed 88.80% resistance against Cefepime in the present study which is not consistent with the results of Zehra et al. [31] Romao et al. [28] and Tripathi et al. [29] who all detected 77.70%, 66%, 71%, 36.27%resistance against Cefepime respectively.The reason in the difference could be because of geographical differences.The fact is antibiotic resistance vary in different regions, environmental conditions and time to time.
Morganella morganii in-vitro MDR-Patteren showed 11% resistance against Cefepime.The finding of the current study is in agreement with the results of Falagas et al. [24], who found 8% resistance against Cefepime.Similarly Xiao-Bo et al. [32] found 14.30% resistance to Cefepime.In this study, the resistance of MDR-M.morganii against Imipenem 66.60% followed by Cefoperazone and Ceftriaxone 55.50% equals while Ceftazidime have 48.10%.These results were quite different from the results of Falagas et al. [24] who showed resistance 80% against Ceftriaxone and 90% against Ceftazidime.Xiao et al. [32] found 16.50% resistance against Imipenem and 5.50% against Amikacin.Similarly, Lee et al. [25] found 19.40% resistance against Imipenem.The difference is due to geographical locations, specimens types and extraction protocols.
In this study in-vitro MDR-P.mirabillis showed 74.07%resistance against Ceftriaxone followed by 59.20% against Cefepime, 44.40% against Ceftazidime, 37.30% against Ceftazidime and 25.90% against Imipenem.These results are in contradiction with the some of the earlier studies conducted by Falagas et al. [24] observed 100% resistance against Cefepime , Ceftazidime and 14.30% against Imipenem in P. mirabillis.Sharma et al. [33] examined 9.60% resistance against Cefoperazone and 4.80% resistance against Cefepime in P. mirabillis.Xiao et al. [32] detected 7.60% resistance against Ceftriaxone, 6.40% against Ceftazidime, 2.40% against Cefepime, 0.60% against Imipenem and 0.30% against Cefoperazone in P. mirabillis.Here also the difference in our results and others are because of sampling size and difference in specimen types.

Conclusion
The present study in-vitro MDR-Patteren S. typhi showed 40.70% resistance against Ceftriaxone, 37.03% Ceftazidime, 25.90% Cefoperazon, 74.07%Imipenem and 3.70% Cefepime.These results are quite in agreement with the findings of Mustaq [34], while different from the results of Nagshetty et al. [26] who showed 6.31% resistance against Ceftriaxone.Kumar et al. [35] found 12.10% resistance against Ceftriaxone.The difference may be due to the overuse of Ceftriaxone in our region.The study of multi-drug resistance is still an unsolved issue and needs further future endures [36] [37].If this scenario is not properly tackled, there is a time coming, when there will be no more medication effect in terms of antibiotic use in order to cure and save human health and life.

Figure 1 .
Figure 1.MRD-Bacteria percent isolated samples from different parts of the body of Patients.

Figure 2 .
Figure 2. Antibiotics percent resistance in multi drug resistant bacteria from isolated samples.

Table 1 .
Distribution of MDR-bacteria in relation to gender and age wise in present study.

Table 2 .
Clinical isolates of gram negative non-fermenters bacteria from different sites in hospitals.
* n for numbers: % for percentage.

Table 3 .
Antibiotic resistance percentage of gram negative non fermented bacteria (n = 81) isolated from different clinical samples.