Antibiogram Profile and Detection of Resistance Genes in Pseudomonas aeruginosa Recovered from Hospital Wastewater Effluent

The nosocomial pathogen Pseudomonas aeruginosa (P. aeruginosa) is characterized by increased prevalence in hospital wastewater and is a public health concern. Untreated wastewater severely challenges human health when discharged into nearby aquatic ecosystems. The antibiogram profiles and resistance genes of P. aeruginosa were evaluated in this study. Wastewater effluents were obtained from a hospital within a six-month sampling period. After the samples were processed and analysed, P. aeruginosa was identified by polymerase chain reaction (PCR) by amplifying OprI and OprL genes. The Kirby–Bauer diffusion technique was employed to check the susceptibility profiles of P. aeruginosa which were further interpreted using CLSI guidelines. A total of 21 resistance genes were investigated among the isolates. The sum of 81 positive P. aeruginosa were isolated in this study. This study’s mean count of Pseudomonas aeruginosa ranged from 2.4 × 105 to 6.5 × 105 CFU/mL. A significant proportion of the isolates were susceptible to imipenem (93%), tobramycin (85%), norfloxacin (85%), aztreonam (70%), ciprofloxacin (51%), meropenem (47%), levofloxacin (43%), and gentamicin (40%). Meanwhile, a low susceptibility was recorded for amikacin and ceftazidime. The overall multiple antibiotics resistance index (MARI) ranged from 0.3 to 0.9, with 75% of the multidrug-resistant isolates. The assessment of β-lactam-resistant genes revealed blaOXA-1 (3.7%) and blaSHV (2.4%). The frequency of carbapenem genes was 6.6% for blaIMP, 6.6% for blaKPC, 6.6% for blaoxa-48, 2.2% for blaNDM-1, 2.2% for blaGES, and 2.2% for blaVIM. Of the aminoglycoside genes screened, 8.6% harboured strA, 11.5% harboured aadA, and 1.5% harboured aph(3)-Ia(aphA1). Only one non-β-lactamase gene (qnrA) was detected, with a prevalence of 4.9%. The findings of this study revealed a high prevalence of multidrug-resistant P. aeruginosa and resistance determinants potentially posing environmental health risks.


Introduction
Pseudomonas aeruginosa (P.aeruginosa) is a common opportunistic pathogen with increased mortality and morbidity in immunocompromised individuals.It is commonly associated with nosocomial infection, high mortality and morbidity rates, and high treatment costs [1].One of the primary contributors to mortality from Gram-negative septicaemia is Pseudomonas aeruginosa infection, which colonizes the lower respiratory and gastrointestinal tracts.Antibiotic-resistant pathogens of hospital origin pose an increased burden on the healthcare system and worldwide financial costs [2].ESKAPE pathogens are the leading cause of hospital-acquired infections globally, with limited alternative treatment options due to their resistance mechanisms.The rate of collapse due to antibiotics administration, either in single or combination usage, increases the possibility of antibiotics with little or no effectiveness in the future [3,4].
Antibiotic-resistant P. aeruginosa is channelled into the natural environment through sewage disposals, fresh water, open ocean, and hospital effluents.Hospital effluents are one of the most common reservoirs of P. aeruginosa, characterized by inherent multidrug resistance with several strains resistant to various antibiotics [5].Treating infectious diseases using antibiotics in hospitals promotes the spread of antibiotic-resistant bacteria in the environment due to antibiotic discharge and pharmaceutical wastes and chemicals [6].Therefore, resistance is acquired through direct contact with sewage and effluents associated with resistant P. aeruginosa isolates [7].Multidrug-resistant P. aeruginosa (MDRPA) discharge is, thus, increasingly perceived as a common driver in the global spread of antimicrobial resistance and calls for urgent public health action [8].
P. aeruginosa has been known to resist numerous antibiotics due to its outer membrane's low permeability, multidrug efflux pumps, and biofilm formation [9].The burden of resistant genes of distinct bacterial pathogens in hospital wastewater is a public health concern [6].Mismanagement and abuse of antibiotics by humans stimulate the rapid escalation of genes associated with resistance in hospital wastewater, resulting in the rise of drug-resistant organisms in hospitals [10].One of the mechanisms of P. aeruginosa's resistance to antimicrobials is the production of β-lactamase enzymes like extended-spectrum β-lactamases (ESBLs) and AmpC and Metallo β-lactamases (MBLs) [11].ESBLs hydrolyse penicillin, cephalosporins, and aztreonam.Meanwhile, AmpC β-lactamases hydrolyse cephalosporins and cephamycins and resist inhibition by clavulanate, sulbactam, and tazobactam.MBLs hydrolyse carbapenems and other β-lactams [12].Carbapenems are effective against penicillin and cephalosporin-resistant Gram-negative bacteria [13].The most important mechanism of carbapenem resistance in P. aeruginosa is the acquisition of genes encoding MBLs [14].Class A ESBL enzymes such as blaVEB, blaSHV, bla CTX-M , bla PER , bla GES , and bla TEM are common among P. aeruginosa and bla OXA-1 -type ESBL enzymes of class D [15].The acquisition of new resistance genes by P. aeruginosa is intensified by its distribution into aquatic ecosystems [16].Metagenomic investigation of untreated sewage used to characterize the bacterial resistome revealed significant changes in the diversity and abundance of antimicrobial resistance (AMR) genes [17,18].
There is a knowledge gap regarding the health risks associated with P. aeruginosa in hospital wastewater in South Africa.However, there is a need to evaluate the susceptibility patterns of hospital effluents before discharge to curb the increased health risk.As a result, this research aims to assess the prevalence, antibiogram profile, and burden of resistance determinants of P. aeruginosa isolated from hospital wastewater in South Africa.

Colony Enumeration and Prevalence of P. aeruginosa
For the six-month sampling period (April-September), the average colony counts of P. aeruginosa ranged from 2.4 × 10 5 to 6.5 × 10 5 CFU/mL.Out of 144 presumptive isolates, 81 (56.25%) were confirmed by PCR assay as P. aeruginosa.

MDR and MARI Analysis
More than half-61 (75%)-of the phenotypically resistant P. aeruginosa isolates w resistant to three or more antibiotics classes, hence their classification as multidrugsistant.The multiple antibiotics resistance index (MARI) of P. aeruginosa isolates rang from 0.3 to 0.9.The MAR indices are shown in Table 1.

No. of Antibiotics MAR Phenotypes
No. of Phenotypes MAR Index

MDR and MARI Analysis
More than half-61 (75%)-of the phenotypically resistant P. aeruginosa isolates were resistant to three or more antibiotics classes, hence their classification as multidrug-resistant.The multiple antibiotics resistance index (MARI) of P. aeruginosa isolates ranged from 0.3 to 0.9.The MAR indices are shown in Table 1.
Table 1.MAR indices of resistant P. aeruginosa isolates.

No. of Antibiotics MAR Phenotypes
No. of Phenotypes MAR Index

Discussion
The widespread release of antibiotics and resistant bacteria into the environment through hospital effluents is an alarming public health concern.Hospital wastewater effluents serve as a primary source of antibiotics spread, hence the presence of antibiotics in various settings, such as domestic and hospital sewage [19].These wastewater effluents are not effectively treated before they are released into nearby rivers.The importance of water bodies as a route for spreading bacteria has been acknowledged, and this spread is often associated with effluents from hospitals, animal farmland, and municipal sewage systems [20].Contaminants found in hospital effluents usually resist water treatment, thereby being disseminated into nearby aquatic environments [21].Antibiotics, major contaminants in wastewater effluents, are released into marine environments, disrupting ecosystems and posing a higher health risk when residents depend on rivers for irrigation, drinking, and recreational activities.Antibiotics resistance is a global menace to humans and animals [22].Our investigation's amplification of the OprI and OprL genes provided evidence of P. aeruginosa.The outer membrane lipoprotein OprI appears in two primary forms: a bound form, connected to a cell wall peptidoglycan, and a free form, lacking anchoring.The outer membrane protein OprL in P. aeruginosa is essential in the organism's resistance to antibiotics and antiseptics [23].The resistance stress of nosocomial pathogens such as P. aeruginosa isolated from untreated hospital wastewater was assessed in this study by analysing the antibiogram profile and resistance genes.
This study revealed the burden of P. aeruginosa in the study area with a prevalence of 56.25% (81/144).The average prevalence rate of P. aeruginosa recovered in this study could be due to the nutritional value of the hospital effluents and increased hospitalrelated infections.The prevalence of P. aeruginosa reported in this study is similar to that reported in another study where 72 P. aeruginosa isolates were obtained from hospital effluents [6].Other studies have also reported reduced incidences of 26.04%, 20.83%, 14.58%, 18.75%, and 19.79% [7].This study's mean count of Pseudomonas aeruginosa ranged from 2.4 × 10 5 to 6.5 × 10 5 CFU/mL.During the sampling period, there was an observable progression in Pseudomonas aeruginosa count in May and July, while April, June, August, and September experienced a steady decline.Another report recorded a similar mean Pseudomonas aeruginosa population density ranging from 1.7 × 10 5 -6.1 × 10 5 CFU/mL [7].
Most bacteria of clinical and environmental origin are mainly inhibited by antibiotics, whose consequence is severe therapy frustration.The confirmed isolates in our study revealed high sensitivity to the following classes of antibiotics: fluoroquinolones, carbapenems, aminoglycosides, and monobactams.The most effective drug in this study is imipenem (96%), a carbapenem with an occurrence rate of 96%.Carbapenem is a β-lactam antibiotic that has demonstrated efficacy for extended-spectrum β-lactamase-producing bacteria.Carbapenems are regarded as a last-resort antibiotic for treating illnesses brought on by multidrug-resistant Gram-negative bacteria.Carbapenems inhibit the peptidase domain of PBPs and also hinder peptide cross-linking.The capacity of carbapenems to bind to various PBPs is crucial to their efficiency.A similar high susceptibility of Pseudomonas aeruginosa against imipenem was reported in other studies [7,24].Meanwhile, meropenem, also under the carbapenem class of antibiotics, showed a lower sensitivity rate of 47%.Regarding the fluoroquinolone class of antibiotics, 85%, 51%, and 43% of confirmed Pseudomonas aeruginosa isolates were susceptible to norfloxacin, ciprofloxacin, and levofloxacin.The fluoroquinolones inhibit type II DNA topoisomerases (gyrases) required for synthesizing bacterial mRNAs (transcription) and DNA replication.A study in South Africa also reported a similar sensitivity rate of 89% to norfloxacin [25].The aminoglycosides, which include tobramycin, gentamicin, and amikacin, exhibited high sensitivity to the isolates.Aminoglycosides are known to reduce the growth of bacteria by blocking protein synthesis with high affinity to a ribosomal RNA active site in the 30S ribosome.A similar study reported a sensitivity rate of 93% against tobramycin [25].Monobactams act mainly by inhibiting penicillin-binding protein 3 (PBP3), interfering with peptidoglycan synthesis and cell division.Aztreonam, a monobactam, was sensitive in this study in 70% of the Pseudomonas aeruginosa isolates.Ceftazidime and amikacin recorded a low sensitivity pattern among the isolates.Antibiotics resistance is a severe challenge that calls for urgent attention.The resistance of P. aeruginosa to ceftazidime is consistent with another survey [26].Intriguingly, none of the isolates were resistant to all tested antibiotics.MARI measures the health risks resulting from drug resistance spread in a given environment.The MARI range of 0.3-0.9observed in this study is greater than the limit of >0.2, which shows that the isolates emanated from habitats harbouring numerous antibiotics.The value also indicates that chromosomes or plasmids enable antibiotics' mechanisms.The MARI result in this study implies the environmental burden of antibiotic use and potential antibiotic pressure [7].A similar MARI ranging from 0.33-0.89was reported in a previous study [7].Findings from P. aeruginosa isolated from abattoirs also had a MARI range of 0.4 to 0.8, similar to this study [9].In this study, about 75% of the isolates were resistant to three or more classes of antibiotics, hence their classification as multidrug-resistant.Furthermore, among the MDR isolates, 14.8% were resistant to three antibiotics, 11.1% to four antibiotics, 14.8% to five antibiotics, 14.8% to six antibiotics, 12.3% to seven antibiotics, 4.9% to eight antibiotics, and 2.5% to nine antibiotics.Meanwhile, 25% of the P. aeruginosa isolates were resistant to less than three antibiotics.
P. aeruginosa acquires resistance traits by transferring genes between humans, animals, and the environment [27].Production of β-lactamases is one of the resistance mechanisms of β-lactam antibiotics in P. aeruginosa, which inactivates the drugs, thereby mitigating the effect of antibiotics therapy [28].Production of carbapenemase enables resistance to cephalosporins and monobactams.This study screened 21 resistance genes among P. aeruginosa isolates that showed varied resistance rates to the antibiotics tested.The aminoglycoside genes had the highest occurrence in this study, followed closely by the carbapenem genes.Among the isolates resistant to aminoglycosides, 8.6% harboured strA, 11.5% harboured aadA, and 1.5% harboured aph(3)-Ia(aphA1).The carbapenem genes bla IMP , bla KPC , and bla oxa-48 have an exact prevalence of 6.6%; likewise, bla NDM-1 , bla GES , and bla VIM have a common prevalence rate of 2.2%.This study also detected two β-lactamase genes, with bla OXA-1 being the most predominant, with a prevalence rate of 3.7%, alongside bla SHV (2.4%).QnrA is the only quinolone gene isolated in this study, with a rate of 4.9%.This study's high abundance of antimicrobial resistance genes indicates an environmental burden, increasing possible health risks.

Research Area
The study was conducted between April 2021 and September 2021 at Victoria Hospital in Amathole District Municipality (ADM).The hospital is in Alice, a second small town in the Nkonkobe Municipality west of Amathole district (ADM), with an area coverage of 21,117 km 2 and a population of approximately 880,790 people.Victoria Hospital is a provincial government-funded hospital in South Africa and the largest referral hospital for residents with numerous healthcare problems located in Raymond Mhlaba's local municipality in ADM.ADM is situated in the heart of the Eastern Cape Province.It runs from the Fish River mouth on the Sunshine Coast.The Amathole Mountain Range borders ADM to the north.An estimated 72% of the municipality's population lives in rural areas; 46% live in poverty, and 57.5% are out of a job [29].The major economic sectors include community services, finance, manufacturing, trade, transport, and agriculture.The region is one of the indigents in South Africa and comprises mainly rural areas with substandard sanitary facilities.A map showing the sampling site and the neighbouring communities is shown in Figure 2.

Sample Collection
Wastewater effluent was aseptically obtained in triplicates from the sampling location in one-litre sterile glass bottles.An ice box was used to store the samples within 6 h of collection before being transported to the laboratory for analysis.

Isolation of Presumptive P. aeruginosa
A tenfold serial dilution of the wastewater samples was performed.A 100 mL aliquot was filtered through a membrane filter with a pore size of 0.45 µm (Sartorius, Goettingen, Germany) as described by the membrane filtration technique.The filtrate was aseptically placed on a plate with a cetrimide agar surface (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 • C for 24 h.Each sample was analysed in triplicates.Distinct colonies with characteristic pyocyanin and pyoverdine pigments showing blue-green and yellow-green colours were considered presumptive for P. aeruginosa.The population densities of P. aeruginosa on cetrimide agar were counted as colony-forming per 100 mL (CFU/mL).The presumptive P. aeruginosa was purified on nutrient agar and stored for subsequent use.

DNA Extraction
The genomic DNA of presumptive isolates of P. aeruginosa was extracted by a boiling method, as previously described [30].Two (2 mls) of the overnight cultures were placed into sterile Eppendorf tubes (Scientific Specialities, Inc., Lodi, CA, USA) and centrifuged (LASEC, Cape Town, South Africa) for 5 min at 13,500× g rpm.The supernatant was decanted, after which 500 µL of sterile distilled water was added and vortexed for 2 min using VELP, Scientifica.To lyse the cells, the suspension was heated at 100 • C for 10 min with MS2 Dri-Block DB.2A (Techne, London, UK) then placed on ice to cool.Subsequently, the suspension was centrifuged for 5 min at 13,500× g rpm, and the supernatant was carefully decanted into sterile Eppendorf tubes to be used as DNA templates.For further molecular analysis, the DNA templates were stored at −80 • C.

Multiple Antibiotics Resistance Phenotype (MARP) and Multiple Antibiotics Resistance Index (MARI) Analysis
MARP patterns for multidrug-resistant P. aeruginosa isolates were evaluated by indicating the sequence of resistance to individual antibiotics.MARI was also assessed and interpreted for all positive P. aeruginosa isolates, as previously stated [33], MARI was calculated by dividing the number of antimicrobials to which the organisms were resistant (a) by the total number of antimicrobials (b) the organisms were exposed to.A MARI value greater than 0.2 indicates a possible environmental source of contamination due to high exposure to antibiotics, while a MARI value of ≤0.2 denotes minimal exposure to antibiotics usage.

Conclusions
P. aeruginosa is a significant cause of hospital-acquired infection with severe disease risk.Hospital effluents are reservoirs of resistant P. aeruginosa isolates.There is a continuous and rapid spread of resistant P. aeruginosa in hospital effluents, causing unrest in aquatic ecosystems.Our research found that untreated hospital wastewater contained a substantial clinical pathogen (P.aeruginosa), which might potentially be released into the environment.Considering the high sensitivity of some of the isolates, most of the tested antibiotics can be administered for therapeutic purposes, excluding ceftazidime and amikacin.The capacity of P. aeruginosa to resist antimicrobial actions results in its spread and survival in the environment and, hence, its difficult elimination.Drug abuse and selective drug pressure are familiar drivers of antibiotics resistance.This research revealed antibiotic determinants' burden in the study area.These resistance genes allow resistance to antimicrobials and result in unproductive treatment measures.This study is limited by the inability to sample more nosocomial pathogens and perform clonality analysis.Therefore, there is a need for proper surveillance and monitoring of hospital effluent discharge to circumvent the continuous dissemination and proliferation of resistant bacteria.Hospital wastewater should be adequately treated and be within the recommended safe standard before disposal.

Figure 2 .
Figure 2. A map showing the study area.

Table 1 .
MAR indices of resistant P. aeruginosa isolates.

Table 2 .
Distribution of resistance genes among P. aeruginosa isolates.

Table 3 .
Primer sequences for the molecular identification of P. aeruginosa.