Rising prevalence of uropathogenic bacteria from UTI in pregnant women

Abstract

Link to original article: https://www.sciencedirect.com/science/article/pii/S1658361220301621

Objectives

Keywords

Introduction

Urinary tract infections (UTIs) are the most frequently occurring infections affecting the human population. They cause severe complications in millions of individuals annually. Estimates state that over seven million cases of UTI have been reported in emergencies, while around 100,000 cases of nosocomial infections have been reported in healthcare units annually.¹ Infections induced by bacteria that affect even a certain portion of the urinary tract are a major cause of UTI. These include increased urine frequency along with pain and the presence of cloudiness in the urine.² Other symptoms comprise dysuria, cramps in the lower abdominal region, back pain, chills, fever, and general weakness accompanied by nausea and vomiting.³ Generally, urine contains not only bacteria, but also salts, wastes, and fluids. It has been seen that pathogenic bacteria entering and multiplying in the bladder or kidneys eventually become a cause of UTIs.^2,4 Asymptomatic bacteriuria, if left untreated, might cause acute cystitis and also pyelonephritis, which could eventually lead to serious consequences such as prematurity, low birth weight, and increased foetal mortality rates.5, 6, 7

Most commonly, UTI is caused by gram-negative aerobic bacilli that originate in the gastrointestinal tract. Frequent pathogens responsible for UTI include Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Proteus, Pseudomonas, Serratia, and Staphylococcus, which colonise the genito-urinary tract.^6,8, 9, 10, 11 Ahmed et al.⁸ revealed that Klebsiella, Escherichia coli, Pseudomonas, and Proteus spp. are the most influential gram-negative organisms responsible for UTIs. Among gram-positive bacterial pathogenic strains, Staphylococcus and Streptococcus spp. have been found to be the most common bacteria responsible for UTIs.⁵ The main subject of concern to date has been the treatment of UTIs and whether it could decrease maternal or neonatal complications. Studies have revealed that antibiotic regimens have not shown any decrease in these complications and have imposed a huge financial burden on society.¹² Antibiotic resistance occurs when bacteria have or develop the ability to circumvent the mechanism, which drugs use against them.¹³ The resistance of uropathogenic bacteria against antimicrobials is rising globally over time. It is directly dependent on the use and misuse of antimicrobial drugs, along with geographical location. Knowing the consequences of resistance is vital as the varying rates of resistance exert a massive influence on empirical therapies of UTIs.⁷

Microorganisms such as Acinetobacter, Escherichia, and Pseudomonas aeruginosa are notably distinguished due to their intrinsic capability of exposing multidrug resistance (MDR). Microbes that have been treated for several years have been showing resistance to drugs in recent times.¹⁴ Colonisation with MDR bacteria can lead to infection that is more difficult to treat.¹⁵ The conditions are even more challenging with the increasing prevalence of UTI as well as the reduced discovery of new antibiotics.¹⁶ Dammeyer et al.¹⁷ reported a higher risk of colonisation of methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamase-producing E. coli transmitted from mother to child during pregnancy and after childbirth. The MDR in bacteria could be due to various mechanisms of action including antibiotic destruction, modification, alterations, and reduction in antibiotic accumulation due to inhibition of permeability and increased efflux.¹⁸ Genes are transmitted within bacteria by various means, and the most vital means is conjugation. Reduced permeability prevents the antimicrobial agent from entering into the cell wall of bacteria, because of which the intracellular concentration of the antimicrobial agent is reduced.¹⁹ Antibiotic pressure explains the fact that resistance towards antibiotics might arise with the emergence of resistant strains and the killing of susceptible strains. In patients infected with resistant strains, antimicrobial usage may inhibit susceptible strains and sanction resistant strains to multiply.¹⁴ Therefore, the current study aimed to determine the prevalence of UTI and the frequency of MDR uropathogenic bacterial strains in pregnant women in Lahore, Pakistan. These uropathogenic bacterial strains were characterised biochemically and identified using 16S rRNA gene sequencing.

Materials and Methods

A cross-sectional study was conducted from December 2018 to June 2019. Urine samples (n = 80) were collected from secondary care, Hussain Memorial Hospitals, Lahore, Pakistan, and informed consent of participants was recorded on a form that included explicit consent for the collection, storage, and testing of the samples. In this study, pregnant women aged ≥18 years attending the antenatal clinics with symptomatic (dysuria, burning micturition, frequency, urgency, lower abdominal pain, and fever) and asymptomatic UTIs were included. Normally, patients with more than four to five pus cells, estimated through urine microscopy, which serves as a baseline antenatal visit, were investigated to determine the prevalence of UTI and isolation of uropathogenic bacteria. Urine microscopy was performed by taking 10 mL of urine in a test tube that was then centrifuged at 3000 rpm for 10 min. The supernatant was decanted, and a drop of sediment was placed on microscopic slides. Microscopic slides were covered with a coverslip and observed under a light microscope. UTI patients who had taken antibiotics in the last two weeks were not included in the study. Socio-demographic characteristics, including medical and obstetrical history, clinical signs, and obstetric characteristics of the index pregnancy, were collected using a structured questionnaire.

Urine collection and isolation of uropathogenic bacteria

Midstream random voided urine samples (10–15 mL) were collected in sterile disposable containers and transferred to the Microbiology Laboratory, Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan, for bacterial isolation within two to 3 h. Bacterial strains were isolated through serially diluting (10⁻⁵) urine samples and spreading on autoclaved cysteine-lactose-electrolyte-deficient (CLED) agar. Petri plates were incubated at 37 °C for 72 h in an incubator. The resulting colonies were manually counted to record the colony-forming units (CFUs) of bacteria in the urine sample. Distinctly different morphological colonies were purified using the multiple-streak method and preserved in glycerol stock at −20 °C for further culturing and characterisation experiments.

Characterisation of uropathogenic bacteria

For the identification of bacterial genera, strains were characterised through various morphological and biochemical tests as recommended by Hafeez and Aslanzadeh.²⁰ The strains were screened using Gram staining and differentiated into gram-positive and gram-negative by observing whether they were purple- or pink-stained, respectively.²¹ Bacterial morphology was determined through a simple staining method as described by Cappuccino and Sherman.²² The bacterial strains were cultured on mannitol salt agar (MSA); yellow colonies that fermented mannitol and exhibited coagulase and deoxyribonuclease (DNase) activity were considered to be S. aureus.²³ The strains were further cultured on MacConkey agar to differentiate lactose fermenting bacterial strains that appeared in the form of pink colonies.²⁴ The standard microbiological methods for triple sugar iron (TSI), hydrogen sulphide (H₂S), indole production, catalase, oxidase, urease, and citrate utilisation, bile-esculin, coagulase, and DNase tests were performed in triplicate by following the methods of Cappuccino and Sherman.²²

MDR assay

The MDR assay of uropathogenic bacterial strains was performed using the disk diffusion method.²⁵ All identified strains in the Escherichia, Klebsiella, Pseudomonas, Streptococcus, Enterococcus, Staphylococcus, and Proteus genera were tested for MDR. Autoclaved Mueller-Hinton (MH) agar plates (90-mm diameter) were prepared, and the bacterial inoculum of 10⁸ CFU mL⁻¹ was heavily streaked using a sterile cotton swab. For quality control, E. coli strain ATCC 25922 was also run parallel to the antibiotic assay. A total of 28 commercially-available paper antibiotic disks including amikacin (AK), amoxicillin (AML), amoxicillin clavulanate (AMC), ampicillin (AMP), aztreonam (ATM), ceftazidime (CAZ), cephradine (CE), ciprofloxacin (CIP), gentamycin (CN), ceftriaxone (CRO), cefotaxime (CTX), ceforaxime (CXM), doxycycline (DO), cefipime (FEP), fosfomycin (FOS), imipenem (IPM), levofloxacin (LEV), linezolid (LZD), meronem (MEM), moxifloxacin (MXF), nitrofurantoin (NF), novobiocin (NV), penicillin (P), pipemidic acid (PIP), tetracycline (TE), tobramycin (TOB), piperacillin tazobactam (TZP), and vancomycin (VA) of fixed concentration (Oxoid, UK) were placed on the inoculated agar surface and incubated for 24 h at 37 °C. The standard tested antibiotics were selected from the tables provided by the Clinical and Laboratory Standards Institute (CLSI) appropriate for testing Enterobacteriaceae, Pseudomonas, Staphylococci, Enterococci, and Streptococci spp.^26,27 After incubation, the diameter of zones of growth inhibition around the antibiotic disc were measured in millimetres. The diameter of the zone of growth inhibition was interpreted as sensitive, intermediate, or resistant as reported by CLSI.²⁶

Molecular characterisation

The most MDR strains from the Escherichia, Klebsiella, and Pseudomonas genera were selected for identification using 16S rRNA partial gene sequencing. The selected strains were identified using genomic DNA isolation, amplification, sequencing of nucleotides, and phylogenetic analysis. The bacterial biomass was treated with proteinase-K treatment for the isolation of genomic DNA.²⁸ For PCR, 2.5 μL genomic DNA was used. The PCR reaction was performed using primers 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3′ and 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3'. The amplified PCR product was run on 1% agarose gel with GeneRuler 1 kb DNA (Fermentas) to confirm the size of the amplified 16S rRNA and further purified using a PCR purification kit (Favorgen, Taiwan). The amplified PCR products were sequenced using the commercial services of MACROGEN Seoul, Korea (http://macrogen.com/eng/). The resulting nucleotide sequences were blasted on NCBI servers by optimising highly similar sequences (Megablast). The sequences of closely related strain types were selected, and the phylogenetic tree was drawn using MEGA 7.0.14.²⁹

Results

Characterisation of uropathogenic bacterial strains

Prevalence of multidrug resistance uropathogenic bacterial strains

A total of 13 uropathogenic Pseudomonas strains were screened against various antimicrobial drugs to determine their MDR pattern, and the results are depicted in Table 3. Approximately all tested Pseudomonas strains were sensitive to MEM, whereas more than 60% of Pseudomonas strains were sensitive to CAZ, CN, IMP, PIP, and TZP. More than 50% of Pseudomonas strains had MDR patterns against ATM, CIP, and CTX. Three strains each from the Staphylococcus, Streptococcus, and Enterococcus genera, and two from the Proteus genus were evaluated for sensitivity to and resistance against various antimicrobial drugs, and their results are given in Table 4. Among the three strains of Staphylococcus, all strains were resistant to DO, TE, and TOB, and they had intermediate resistance against PIP. All strains of Staphylococcus were sensitive to CIP, CN, LEV, MXF, NF, NV, and VA. In case of Streptococcus (n = 3), all strains were resistant to CE, CIP, DO, LEV, P, and TE. Two strains of Streptococcus were sensitive to FOS, MXF, and NF. All strains of Enterococcus were resistant to CE, CIP, CRO, LEV, P, and VA and were sensitive to LZD. Two Proteus strains were resistant to FOS, NF, and PIP and were sensitive to IMP, MEM, and SCF.

Identification of selected strains using 16S rRNA gene sequencing

Discussion

MDR bacteria can resist the action of antimicrobial drugs, which causes treatment failure of diseases and spread of infections. MDR in bacteria is a natural phenomenon that is extensively promoted in immunosuppressed conditions. It is enhanced due to prolonged drug exposure and the persistence of infections despite therapy. Among infections, UTI is a severe threat in our community, especially to pregnant women.¹ Asymptomatic bacteriuria is a frequently occurring bacterial infection of the urinary tract that requires medical treatment even during pregnancy. Gram-negative and gram-positive bacteria are involved in the occurrence of UTI; however, gram-negative bacteria more commonly cause UTIs.

In the present study, uropathogenic bacterial strains were isolated from 80 pregnant women aged 19–45 years with 78% of participants aged 25–34 years (Table 1). The participants were multigravida (57.5%) and primigravida (42.5%) with UTI in pregnancy as it has repeatedly been reported to be an element for a substantial rise in UTI.³⁰ The association between UTIs and parity is because of the physiological variations that emerge in pregnancy due to UTIs.³¹ Maternal age, parity, and morbid obesity have been formerly witnessed as threat elements for UTI in pregnant women.³² The prevalence of UTIs in our study population was comparable to that in other studies. In our study, we sampled pregnant women, including both urban and rural residents during all trimesters with the majority in the third trimester in the study catchment zone, presenting at antenatal check-ups in Lahore, Pakistan. Sarwar et al.³³ isolated 370 pathogenic bacterial isolates from 520 pregnant female genitalia residing in various areas of Punjab, Pakistan. They reported that 71% of gynaecological infections were caused by E. coli (41.6%), S. aureus (15.4%), and coagulase-negative staphylococci (12.2%). Similarly, Kumarasamy et al.³⁴ isolated Enterobacteriaceae isolates from UTI, pneumonia, and bloodstream infection and reported 239 metallo-β-lactamase 1 producing isolates from India, 37 isolates from the United Kingdom, and 25 isolates from Pakistan, which were found to be carbapenem-resistant. Recently, a study conducted in Bangladesh on UTI in pregnant women reported high-burden bacteriuria (5.7%) in both symptomatic and asymptomatic cases.³⁵ Similarly, Dashtizade et al.³⁶ also reported the prevalence of UTI in Iranian pregnant women. They reported that the population most commonly affected by UTIs were those aged between 21 and 30 years (48.3%), with high school education (38.8%), and who were housewives (90.8%). In the current study, both symptomatic and asymptomatic UTI cases were investigated. Previously, both symptomatic and asymptomatic bacteriuria studies have also been conducted in Bangladesh,³⁷ Ethiopia,³⁸ and Iran.³⁶ Symptomatic bacteriuria, similar to the current study, was also reported in Pennsylvania,³⁹ Iran,⁴⁰ and India.⁴¹

In the present study, uropathogenic bacterial strains were isolated from 80 pregnant women with bacteriuria. A total of 67 uropathogenic isolates underwent a series of biochemical characterisations (Table 2) based on the recommendations of Lennox and Ackerman.⁴² Based on these biochemical characteristics, the uropathogenic bacterial strains were identified at the genus level and grouped into Escherichia, Klebsiella, Pseudomonas, Staphylococcus, Streptococcus, Enterococcus, and Proteus. Similarly, Rave et al.⁴³ also adopted biochemical identification techniques before profiling the antibiotic susceptibility pattern. In addition, the molecular identification of selected uropathogenic bacterial strains was also conducted in this study by performing 16S rRNA partial gene sequencing to verify the correct identification of strains of the bacterial genus. The UTI aetiology in the current study revealed that 81.3% of participants were affected by UTI caused by uropathogenic gram-negative and gram-positive bacterial strains (Table 1, Table 2). In this population, 90% of the isolated uropathogenic strains belonged to gram-negative bacteria. There was a high rate of bacterial isolation from the Escherichia, Klebsiella, and Pseudomonas genera. Similarly, other UTI aetiology studies have reported the predominance of gram-negative bacteria, especially strains of E. coli and Klebsiella spp. Recently, Lee et al.³⁵ reported that 50% of bacteriuria was caused by E. coli and Klebsiella spp. in pregnant women in Bangladesh. They also reported bacteriuria due to staphylococcal (23% of isolates) and Group B streptococcus species (5.3% of isolates).³⁵ Similarly, Majumder et al.⁴⁴ and Haque et al.⁴⁵ reported a predominance of E. coli comprising up to 75% of isolates and Klebsiella species accounting for up to 11% of isolates. Untreated recurrent UTI could be a major cause of foetal and maternal adverse effects such as low birth weight, preterm delivery, cystitis, and pyelonephritis.⁴⁶ Uropathogenic bacteria might enter the urothelium, thus avoiding normal clearance during emptying of the bladder, which later causes recurrent UTI.⁴⁷

Rising antibiotic resistance is a global concern, particularly in low-income countries, including Pakistan. The current study demonstrates the higher rates of antibiotic resistance to common antimicrobial agents for the treatment of UTI in pregnancy. The general pattern was that Escherichia spp. were resistant against AML, PIP, and AMP; Klebsiella spp. were resistant against PIP, AMP, and CTX, and Pseudomonas spp. were resistant against CIP and CTX (Table 3). Other strains from the Staphylococcus, Streptococcus, Enterococcus, and Proteus genera also showed a variable MDR pattern (Table 4). The current study concluded that gram-negative uropathogenic bacterial strains were highly resistant to third-generation cephalosporin antibiotics. Similarly, the World Health Organization (WHO) also reported the resistance of E. coli (68%) and Klebsiella (81%) isolates to third-generation cephalosporin antibiotics.^48,49 Similarly, other relevant studies on MDR in strains of E. coli, Klebsiella, Pseudomonas, and other uropathogenic bacteria have also been reported.^{5,35,36,41,43,}50, 51, 52 The current study revealed that for uncomplicated and complicated bacteriuria, carbapenems should be preferred over fluoroquinolones and cephalosporins. The data from the current study highlights the need for antibiotic stewardship and the development of new effective antimicrobials that are safe for use in pregnancy.

Conclusion

In the present study, the uropathogenic strains of the Escherichia, Klebsiella, and Pseudomonas genera showed significant resistance to PIP, AMP, AML, FEP, and CTX, and less resistance to MEM and TZP. Because of uropathogenic strains with emerging MDR, our conclusion highlights the limited use of antibiotics and recommends physicians to perform culture examination and identify some genetic mechanisms for UTI treatment during pregnancy.

Recommendations

The increase in antibiotic-resistant UTI in pregnant women from Lahore, Pakistan has been observed due to the increased use of antibiotics. National efforts are needed to control MDR UTI in pregnant women in the country by implementing evidence-based actions monitoring UTI treatment. Our study suggests that raising awareness of the judicious use of antibiotics is essential to limit the increase in resistance levels.

Source of funding

The current research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

Ethical approval for the protocol of the study was granted by the Institute of Molecular Biology and Biotechnology at The University of Lahore, Pakistan, with reference number IMBB-734 and dated 04-10-2018.

Authors' contributions

UA-Conducted research, provided research materials, and collected and organised data. MZM conceptualised and supervised the study, analysed and interpreted the data, and wrote the initial and final draft of the article. AM provided research materials and logistic support. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.