Research Article

Emerging Resistance in Gram Negative Bacteria Isolated from Clinical Samples in Lahore, Pakistan: A Current Epilogue

Mehwish Saleem Khan* and Sumama Farooq

Pathology Lab Sir Ganga Ram Hospital, Lahore, Microbiology Lab Islamia College For Women Cooper Road, Lahore, Inmol Hospital, Lahore.

Received | December 24, 2021; Accepted | March 16, 2022; Published | June 25, 2022

*Correspondence | Mehwish Saleem Khan, Pathology Lab Sir Ganga Ram Hospital, Lahore, Microbiology Lab Islamia College For Women Cooper Road, Lahore, Inmol Hospital, Lahore; Email: [email protected]

Citation | Saleem, M. and S. Farooq. 2022. Emerging resistance in gram negative bacteria isolated from clinical samples in lahore, pakistan: A current epilogue. Journal of Innovative Sciences, 8(1): 53-59.

DOI | https://dx.doi.org/10.17582/journal.jis/2022/8.1.53.59

Keywords | Antibiotics, Antibiotic resistance, Gram-negative bacteria, E. coli bacteremia, Urinary tract infection

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction

Resistance in bacteria is rising to dangerously high levels all over the world. New resistance mechanisms are emerging, we are heading for a post-antibiotic era, in which common infections and minor injuries can once again kill (Scott et al., 2016). Antibiotics have played a vital role in medicine and surgery and have saved billions of lives all around the world (Rossolini et al., 2014). Benefits of antibiotics are limited as compared to harmful effects due to increasing level of resistance in microbes against all available antibiotics which is an alarming situation (Ventola, 2015).

Intrinsic resistance (Blair et al., 2015) and acquired resistance, on the other hand result in horizontal gene transfer (Magiorakos et al., 2012). Direct transfer of DNA through transformation (Hu et al., 2017) leads to chronic infections because heavy dose is required to treat infection (Ribeiro et al., 2016). ß-lactam drugs occupy a special place among antibiotics. Beyond the relative ease of dosing them and their usually good or acceptable tissue distribution (Gootz, 2010). Most important causative agent of urinary tract infection is E. coli (Gupta et al., 2011) due to considerable resistance against variety of antibiotics like tetracycline, ampicillin, fluoroquinolones and cephalosporin (Welch, 2017).

Klebsiella is one of the prominent Genus of Enterobacteriaceae among them K. pneumoniae is the most prominent microbe (Nizet and Klein, 2011) responsible for high rate of causalities (Perez and Van Duin, 2013). P.mirabilis and P.vulgaris has stamina of transformation throughout life (Drzewiecka, 2016). Presence of enzymes AmpC and β lactamases (Bush and Jacoby, 2010). Several studies have revealed that P. mirabilis that produce AmpC β-lactamases and ESBL can cause outbreaks in developing and developed countries (Nakano et al., 2012). Even amino glycosides, carbapenems, cephalosporins and fluoroquinolones resistant strains are reported from different countries which is an alarming situation (Moradali et al., 2017).

1.1 Aims of study

Isolation and identification of infection causing bacteria. Detection of antibiotic resistant bacteria with antibiotic susceptibility profile. Characterization of bacteria with morphological and biochemical tests.

2. Materials and Methods

The present study was conducted at Sir Ganga Ram Hospital in order to study the resistance pattern in gram-negative bacteria.

2.1 Collection of clinical samples

Total 60 clinical samples of blood, urine, pus and sputum were obtained from patients. These samples were collected in sterile containers in order to prevent contaminations. Number, date and time were mentioned at containers to prevent mixing of samples. Media plates of Blood agar, nutrient agar, MacConkey agar and agar were prepared to grow bacteria on them. Before preparation of these media, all the provisions were kept at incubator to get sterility. Sputum and Pus samples were cultured on Blood agar, MacConkey and nutrient agar. While urine samples were cultured on CLED agar. These culture Media were kept in incubator at 37 °C for 24 hours. Smear was prepared from individual purified colony on glass slide. Smear was heat fixed.

2.2 Gram staining

Gram staining was performed to isolate gram negative bacteria from gram-positive. Smear was flooded with Primary stain crystal violet for one minute. Excessive stain was removed with help of blotting paper. Then smear was washed with tap water. Then Gram iodine was poured on smear and allowed it to stand for one minute. After it, smear was flooded with decolorizing agent (95% ethanol) for 1 minute. At the end of staining a counter stain safranine was poured over the smear for 30-60 seconds. Slides were dried in air and then examined under the compound microscope. Gram positive bacteria retained colour of crystal violet while gram-negative bacteria appeared reddish as they retained the color of counter stain safranine.

2.3 Identification of bacteria

Bacteria were identified on the basis of morphological, colonial and biochemical characterizations. In order to study resistance pattern of gram-negative bacteria disc diffusion method was performed. For antimicrobial susceptibility test Muller Hinton media was used. A pure colony of bacteria was picked with the help of sterile loop and inoculated in saline solution. A sterile swab was used to pour bacterial sample on Muller Hinton media.

2.4 Antibiotic susceptibility test

Antibiotic discs of Amikacin, ampicillin, Gentamycin, ceftazidime and meropenem were placed over the media and were kept for 24 hours in incubator at 37 oC. A ruler was used to measure zone of inhibition. Then resistance patterns of different bacteria against number of antibiotics were observed.

3. Results and Discussion

3.1 Prevalence of clinical samples among gender

The infection rate was more prevalent in females as compared to males. Majority of samples n=33(55%) were Female collected from females and n=27(45%) samples were obtained from males. Females are more prone to bacterial infections as compared to males due to anatomical, physiological, behavioral and hormonal imbalance. UTIs are more common in females as compared to males due to anatomical characteristics of urethra and its close proximity to anus. This shows that females are more prone to bacterial infections.

 

The prevalence of different clinical samples is explained in Figure 2. Higher number of samples were of blood n=20(33%) as most people were suffering from bacteremia. Blood samples were followed by urine samples n=18(30%). Urine samples were collected from patients suffering from infections of urinary tract.

 

Pus samples n=12(20%) were obtained from wounds. While minimum number of samples were of sputum n=10(17%).

3.2 Prevalence of bacterial isolates in clinical samples

Data in Figure 3 illustrates the prevalence of different bacterial isolates in clinical samples. E. coli was most prevalent (n=30) in clinical samples. As it is commensal of gastrointestinal tract and is present everywhere in environment so it is more prevalent in clinical samples. Pseudomonas aeruginosa was second most abundant (n=13) isolate in clinical samples followed by Klebsiella spp. (n=12) and Proteus spp.(n=5). These isolates were obtained from body fluids such as blood, urine, pus and sputum.

 

The resistance patterns of different gram negative bacteria obtained from clinical samples were observed against most zcommonly used antibiotics. Total six antibiotics were used, which are most commonly used to treat bacterial infections. Different pathogens have shown different resistance patterns against these antibiotics Amoxicillin (AMX), Amikacin (AMK), Ciproflaxin (CIP), Ceftazidime (CAZ) Gentamycin (GEN) and Meropenem (MEM) were used.

 

Due to abundance of multi-drug resistant microbes, it has become difficult to treat the bacterial infections as very few antibiotics have left as option of chemotherapeutic agents (Cerceo et al., 2016). In present study infection rate was most prevalent in females (55%) as compared to males (45%). The occurrence of UTIs varies according to different ages and genders, but its frequency of occurrence is higher in female as compared to male, which is due to multiple reasons for example anatomy of urethra, its occurrence near the anus, and different hormonal balance in female .UTIs are more prevalent in female especially in pregnancy duration (John et al., 2016). Females are affected from UTIs in gestation period, and it leads to abortion, underdevelopment of infants and premature births. If it is treated at early stage it is beneficial for mother and fetus, but if it is not treated early it may have severe outcomes (Hannan et al., 2012).

The Prevalence of UTIs also varies according to age which demonstrates that age is one risk factor for UTI. Older females are more affected from UTI as compared to young ones due to degeneration of urogenital tract and impairment of vagina after menopause. Due to these reasons the pH of vagina increases and number of vaginal Lactobacillus decrease which allow the gram-negative bacteria to settle down and increase in number and act as uropathogens, so UTI is more prevalent in older females (Nicolle, 2016).

Urine samples n=18(30%) were collected from patients suffering from infections of urinary tract. One of the most prevalent clinical complications is Urinary tract infections (UTIs), it includes the infections of kidneys, ureters, urinary bladder, and urethra (Haider et al., 2010).

In present study n=12(20%) pus samples were obtained from wounds. Wounds are the reservoir of bacteria as they provide nutrition, temperature and humidity to them thus allow bacteria to divide rapidly and form the colonies at the infected site. Wounds are produced as a result of cuts on skin, operations cuts, accidents, some diseases such as Diabetes. The skin act as first line of defense in body but when wounds occur this is destroyed which allows bacteria to invade and cause infections. That’s why wound infections are responsible for variety of infections, longer hospital stays, burden on economy and causalities all around the world. The prominent gram negative bacteria isolated from clinical samples are members of family Enterobacteriaceae and pseudomonas aeruginosa (Sisay et al., 2019).

Sputum samples n=10(17%) were collected from patients suffering from respiratory tract infections especially Pneumonia. One of the common hospital-acquired infections is Hospital-acquired Pneumonia (HAP) which results in longer stays in hospitals, enhanced encumbrance, and high causality rate globally. HAP is the second notorious hospital acquired infection in United States. HAP is caused by bacteria, so it becomes essential to study these bacteria which are causative agents of it. Gram-negative bacteria are prominent cause of pneumonia. HAP results in prolonged hospital stays, economic burden, and causalities.

E.coli was most prevalent isolate (50%) in clinical samples. E. coli isolates obtained from clinical samples were resistant against Amoxicillin, Ceftazidime, Gentamycin, Ciproflaxin and Meropenem. The resistance recorded in these isolates was much greater than the previous studies conducted by other researchers. The study has confirmed that antibiotic resistance is continuously increasing in isolates collected from patients suffering from gram-negative bacterial infections (Olorunmola et al., 2013).

Pseudomonas aeruginosa was second most abundant isolate (22%) in clinical samples. Pseudomonas aeruginosa is widely distributed in all natural environments. This pathogen is responsible for urinary tract infections, blood born infections and severe form of pneumonia. This microbe can exist in two forms in environments either in free swimming form in water bodies or it forms the biofilms. But in special conditions it loses its mobility, comes to surface of water bodies and becomes non-motile (Khan et al., 2010).

While Klebsiella shows maximum resistance towards Cefotaxime and Gentamycin. In a study conducted in India which showed that majority of Klebsiella is sensitive towards Amikacin. As has proven from this study that Klebsiella is 98% sensitive towards Amikacin. The reason of this susceptibility is that this medicine is not commonly used to treat the infections rather it is rarely used in case of infections (Sarathbabu et al., 2012). A study which was conducted in Jordan has confirmed that Klebsiella is least resistant towards one antibiotic (Alsohaili et al., 2015).

In present study Proteus (8%) was present in least numbers. Proteus showed maximum sensitivity towards Amikacin and this antibiotic is used to treat the infections. In another study conducted in Saudi Arabia has same results were obtained and its proven that majority of this pathogen is resistant towards Ampicillin. The results of present study have shown that this pathogen shows resistance towards Amoxicillin (Bahashwan and Shafey, 2013).

Conclusions and Recommendations

Multidrug resistant microbes are emerging rapidly in clinical environments and natural settings. Females are more infected with pathogenic bacteria than males. This is because of anatomical, hormonal, behavioral and physiological differences in two genders. Now a days bacteria are resistant against most commonly used antibiotics. Inappropriate and extensive use of antibiotics leads to emergence of multidrug resistance in microbes.

Novelty Statement

Monthly collection of resistant bacteria according to gender and clinical samples is of paramount importance. Identification of these strains help to design strategies to curtail the emergence and spread of resistance and to devise innovative therapeutic approaches against multidrug-resistant organisms.

Author’s Contribution

Mehwish Saleem khan: Performed experiment, wrote paper.

Sumama Farooq: Edited the paper.

Conflict of interest

The authors have declared no conflict of interest.

References

Alsohaili, S.A., Alharahsheh, M.H., Almshagbeh, M.A., Alkhawaldeh, R.A., and ALkhawaldeh, W.M., 2015. Bacterial pathogen in urinary tract infection and antibiotic resistance patteern in Zaraqa-Jordan. European Scientific Journal. 11(12): 171-177. https://eujournal.org/index.php/esj/article/view/5473

Bahashwan, S.A., and El-Shafey, H.M., 2013. Antimicrobial resistance patterns of Proteus isolates from clinical specimens. European Scientific Journal, 9(27): 188-202. https://www.eujournal.org/index.php/esj/article/view/1819

Blair, J.M., Webber, M.A., Baylay, A.J., Ogbolu, D.O., and Piddock, L.J., 2015. Molecular mechanisms of antibiotic resistance. Nature Reviews Microbiology. 13(1): 42-51. https://www.nature.com/articles/nrmicro3380, https://doi.org/10.1038/nrmicro3380

Bush, K., and Jacoby, G.A., 2010. Updated functional classification of β-lactamases. Antimicrobial Agents and Chemotherapy, 54(3): 969-976. https://aac.asm.org/content/54/3/969, https://doi.org/10.1128/AAC.01009-09

Cerceo, E., Deitelzweig, S.B., Sherman, B.M., and Amin, A.N., 2016. Multidrug-resistant gram- negative bacterial infections in the hospital setting: Overview, implications for clinical practice, and emerging treatment options. Microbial Drug Resistance. 22(5): 412-431.https://pubmed.ncbi.nlm.nih.gov/26866778/, https://doi.org/10.1089/mdr.2015.0220

Drzewiecka, D., 2016. Significance and roles of Proteus spp. bacteria in natural environments. Microbial Ecology. 72(4): 741-758. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080321/, https://doi.org/10.1007/s00248-015-0720-6

Wang, Y.H. and Zhang, T.T., 2019. Factors influencing mortality in hospital-acquired pneumonia caused by Gram-negative bacteria in China. Journal of Infection and Public Health. 12(5): 630-633. https://pubmed.ncbi.nlm.nih.gov/30824328/, https://doi.org/10.1016/j.jiph.2019.02.014

Gootz, T.D., 2010. The global problem of antibiotic resistance. Critical Reviews™ in Immunology, 30(1): 79-93. https://pubmed.ncbi.nlm.nih.gov/20370622/, https://doi.org/10.1615/CritRevImmunol.v30.i1.60

Gupta, K., Hooton, T.M., Naber, K.G., Wullt, B., Colgan, R., Miller, L.G., Moran, G.J., Nicolle, L.E., Raz, R., Schaeffer, A.J. and Soper, D.E., 2011. Effects of antibiotics resistance. Clinical Infectious Diseases, 52(5): 103-120. https://pubmed.ncbi.nlm.nih.gov/21292654/, https://doi.org/10.1093/cid/cir102

Hannan, T.J., Totsika, M., Mansfield, K.J., Moore, K.H., Schembri, M.A., and Hultgren, S.J., 2012. Host–pathoge. European Scientific Journal, 52(4): 1-230.

Haider, G., Zehra, N., Munir, A.A., and Haider, A., 2010. Risk factors of urinary tract infection in pregnancy. The Journal of the Pakistan Medical Association, 60(3): 1-213. https://pubmed.ncbi.nlm.nih.gov/20225781/

Hu, Y., Gao, G.F., and Zhu, B., 2017. The antibiotic resistome: Gene flow in environments, animals and human beings. Frontiers of Medicine, 11 (2): 161-168. https://pubmed.ncbi.nlm.nih.gov/28500429/, https://doi.org/10.1007/s11684-017-0531-x

John, A.S., Mboto, C.I., and Agbo, B., 2016. A review on the prevalence and predisposing factors responsible for urinary tract infection among adults. European Journal of Experimental Biology, 6(4): 7-11. https://www.researchgate.net/publication/303651684_A_review_on_the_adults

Khan, W., Bernier, S.P., Kuchma, S.L., Hammond, J.H., Hasan, F., and O’Toole, G.A., 2010. Aminoglycoside resistance of Pseudomonas aeruginosa biofilms modulated by extracellular polysaccharide. International Microbiology: The Official Journal of the Spanish Society for Microbiology. 13(4): 207-212. https://pubmed.ncbi.nlm.nih.gov/21404215/

Magirokas, M.E., Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Olsson- Liljequist, B. and Paterson, D.L., 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, 18(3): 268-281. https://pubmed.ncbi.nlm.nih.gov/21793988/, https://doi.org/10.1111/j.1469-0691.2011.03570.x

Moradali, M.F., Ghods, S., and Rehm, B.H., 2017. Pseudomonas aeruginosa lifestyle: A paradigm for adaptation, survival, and persistence. Frontiers in Cellular and Infection Microbiology, 7(1): 1-39. https://www.frontiersin.org/articles/10.3389/fcimb.2017.00039/full, https://doi.org/10.3389/fcimb.2017.00039

Nakano, R., Nakano, A., Abe, M., Inoue, M., and Okamoto, R., 2012. Regional outbreak of CTX-M-2 β-lactamase-producing Proteus mirabilis in Japan. Journal of Medical Microbiolog, 61(12): 1727-1735. https://pubmed.ncbi.nlm.nih.gov/22935848/, https://doi.org/10.1099/jmm.0.049726-0

Nicolle, L.E., 2016. Urinary tract infections in the older adult. Clinics in Geriatric Medicine, 32(3): 523- 538. https://doi.org/10.1016/j.cger.2016.03.002

Nizet, V., and Klein, J.O., 2011. Bacterial sepsis and meningitis. Infectious Diseases of the Fetus and Newborn Infant, 7(1): 222-275. https://doi.org/10.1016/B978-1-4160-6400-8.00006-7

Olorunmola, F.O., Kolawole, D.O., and Lamikanra, A., 2013. Antibiotic Resistance and Virulence Properties in Escherichia coli Strains from Cases of Urinary Tract Infections. African Journal of Infectious Diseases. 7(1): 1-7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647523/, https://doi.org/10.4314/ajid.v7i1.1

Perez, F. and Van Duin, D., 2013. Carbapenem-resistant Enterobacteriaceae: A menace to our most vulnerable patients. Cleveland Clinic Journal of Medicine, 80(4): 225-233. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960994/, https://doi.org/10.3949/ccjm.80a.12182

Ribeiro, S.M., Felício, M.R., Boas, E.V., Gonçalves, S., Costa, F.F., Samy, R.P., Santos, N.C. and Franco, O.L., 2016. New frontiers for anti-biofilm drug development. Pharmacology and Therapeutics, 160: 133-144. https://pubmed.ncbi.nlm.nih.gov/26896562/, https://doi.org/10.1016/j.pharmthera.2016.02.006

Rossolini, G. M., Arena, F., Pecile, P., and Pollini, S., 2014. Update on the antibiotic resistance crisis. Current Opinion in Pharmacology, 18(1): 56-60. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253159/, https://doi.org/10.1016/j.coph.2014.09.006

Sarathbabu, R., Ramani, T., Rao, K.B., and Panda, S., 2012. Antibiotic susceptibility pattern of Klebsiella pneumoniae isolated from sputum urine and pus samples. IOSR Journal of Pharmaceutical and Biological Science. 1(2): 04-09. http://www.iosrjournals.org/iosr-jpbs/papers/vol1-issue2/I0120409.pdf?id=4591, https://doi.org/10.9790/3008-0120409

Scheffler, R., Colmer, S., Tynan, H., Demain, A., and Gullo, V., 2013. Antimicrobials, drug discovery, and genome mining. Applied Microbiology and Biotechnology. 97(3): 969-978. https://pubmed.ncbi.nlm.nih.gov/23233204/, https://doi.org/10.1007/s00253-012-4609-8

Scott, G.I., Porter, D.E., Norman, R.S., Scott, C.H., Uyaguari-Diaz, M.I., Maruya, K.A., Weisberg, S.B., Fulton, M.H., Wirth, E.F., Moore, J. and Pennington, P.L., 2016. Antibiotics as CECs: an overview of the hazards posed by antibiotics and antibiotic resistance. Frontiers in Marine Science, 3(1): 1-24. https://core.ac.uk/download/pdf/82883165.pdf, https://doi.org/10.3389/fmars.2016.00024

Sisay, M., Worku, T., and Edessa, D., 2019. Microbial epidemiology and antimicrobial resistance patterns of wound infection in Ethiopia. a meta- analysis of laboratory-based cross- sectional studies. BMC Pharmacology and Toxicology, 20(1): 1-35. https://doi.org/10.1186/s40360-019-0315-9

Ventola, C.L., 2015. The antibiotic resistance crisis: Part 1: Causes and threats. Pharmacy and Therapeutics, 40(4): 270-277. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378521/

Welch, R.A., 2017. Uropathogenic Escherichia coli-Associated Exotoxin. Molecular Pathogenesis and Clinical Management, 4(3): 263-276. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920080/, https://doi.org/10.1128/9781555817404.ch13