Frequency of Fecal Carriage of ESBL Resistance Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates from Cancer Patients at Laquintinie Hospital, Douala, Littoral Region, Cameroon

Background. Opportunistic infections are the second cause of death among cancer patients. This study aimed at determining the antimicrobial profile and the prevalence of extended-spectrum beta-lactamase (ESBL)-gene carriage of Pseudomonas aeruginosa isolates among cancer patients at the Douala Laquintinie Hospital, Littoral Region of Cameroon. Between October 2021 and March 2023, 507 study participants were recruited among whom 307 (60.55%) were cancer patients, compared to 200 (39.45%) noncancer patients. Fifty-eight P. aeruginosa isolates were isolated from fecal samples of forty-five cancer patients and thirteen noncancer patients using Cetrimide agar. The antimicrobial resistance profile of the isolates was determined using the Kirby–Bauer disk diffusion method. The polymerase chain reaction was used to detect the presence of extended-spectrum beta-lactamase genes among P. aeruginosa isolates. P. aeruginosa showed significant resistance rates in cancer patients compared to noncancer patients to imipenem, cefotaxime, and ceftazidime, piperacillin-tazobactam, ticarcillin-clavulanic acid, and ciprofloxacin. The multidrug resistance (MDR) rate was significantly (p < 0.05) higher in cancer patients than in noncancer patients. The frequency of beta-lactamase genes in the 58 ESBL-producing P. aeruginosa isolates was determined as 72.41% for blaTEM, 37.93% for blaOXA, 74.14% for blaCTX‐M, and 44.83% for blaSHV genes. The study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing P. aeruginosa with a high rate of MDR among cancer patients. It indicates that regular monitoring and surveillance of ESBL-producing P. aeruginosa among cancer patients are needed to improve the management of patients.


Background
Patients with compromised immune systems including cancer patients on chemotherapy are highly vulnerable to infections.Opportunistic infections are considered the deadliest among patients.Pseudomonas aeruginosa (P.aeruginosa) is considered one of the principal opportunistic microorganisms responsible for infections among immunocompromised patients.Opportunistic infections are the second cause of death among cancer patients [1,2].Infections constitute one of the main causes of mortality and morbidity in patients sufering from cancer due to the difculty of their management caused by the emergence of multidrug-resistant (MDR) Gram-negative bacteria such as bacteria producing beta-lactamases and resistant nonfermentative rods such as P. aeruginosa.Treatment of bacterial infections has diminished over the past decades because of the increasing rate of antibiotic resistance leading to an increased mortality rate and this greatly interferes with anticancer therapy [3].P. aeruginosa is a Gram-negative aerobic bacterium that is an important cause of nosocomial infections such as ulcerative keratitis (usually associated with contact lens wear), otitis externa (primarily in immunocompromised patients, e.g., with cancer, AIDS, and burns and those with diabetes mellitus), and skin and soft tissue infections (including diabetic foot infections) but also infections in plants, domestic, and farm animals [4,5].Hospitalized patients can be colonized by P. aeruginosa upon admission or contract P. aeruginosa during their hospital stay, and P. aeruginosa can be isolated from almost any imaginable source within hospitals [6].Te World Health Organization (WHO) considers P. aeruginosa among the priority pathogens, which require urgent intervention with new antimicrobial drugs because it is a serious threat due to multiresistance to many antibiotics [7].Multidrugresistant P. aeruginosa infections are responsible for high mortality rates due to their intrinsic resistance to several antimicrobial agents [8][9][10][11].Several mechanisms are involved in the resistance of P. aeruginosa including overexpression of the efux pump and acquisition of extendedspectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) [12].Te increased spread of bacterial resistance to antibiotics has been recognized as a global burden, particularly in developing countries [13].Bacterial resistance to antibiotics in developing countries can be attributed to the overuse of antimicrobials in animals for food consumption, uncontrolled consumption by humans, and imperfect prescription of antibiotics by physicians [14].Tis study aimed to determine the antimicrobial profle and the prevalence of ESBL gene carriage of P. aeruginosa isolates among cancer patients at the Douala Laquintinie Hospital, Littoral Region of Cameroon.

Study Area.
Tis cross-sectional epidemiological study was conducted between October 2021 and March 2023 among consented cancer patients and noncancer patients, regardless of their age and gender, who came for consultation at the oncology unit of the Laquintinie Hospital, Douala, Littoral Region of Cameroon.Fifty-eight patients in whom the presence of P. aeruginosa was identifed from cultured fecal samples were included in this study.Patients who were seropositive for the human immunodefciency virus (HIV), patients on antibiotic treatment, and participants with positive serology for hepatitis B and C were excluded from this study.

Data Collection and Sampling
Procedure.Te methodology used in this study is the same as that used by Ngalani et al. (2020) [15].Stool samples were collected from 507 patients who consented to take part in the study and from whom a bacteriological examination was requested.Duplicates were systematically eliminated.In this study, 507 stool samples were collected under aseptic conditions and processed within two hours of reception.Te feces were collected by the patient as soon as they were passed, using a sterile pot supplied by us.Tis consisted of scraping the fecal matter with the spatula provided and placing it in a sterile pot by the patient.Patients washed their hands thoroughly with soap and water before collection.Any remaining feces were fushed down the toilet.Te sample was returned to the laboratory as soon as possible for immediate microbiological analysis.

Isolation and Identifcation of Pseudomonas aeruginosa.
Stool samples were cultured on Cetrimide agar plates, which are a diferential and selective culture medium for isolation and identifcation of P. aeruginosa and incubated at 37 °C for 24 h.All colonies from the primary culture were purifed by subculturing onto freshly prepared Muller-Hinton agar medium and incubated at 37 °C for 24-48 h.P. aeruginosa was identifed due to its characteristic production of pyocyanin, a blue, water-soluble, nonfuorescent phenazine pigment, coupled with its colonial morphology and the characteristic grape-like odor of aminoacetophenone.We used colony-forming units (CFUs) to count the bacterial density in these samples.Further identifcation was performed using a series of biochemical tests including Gramstaining, an oxidase test, and observation of pyocyanin or pyoverdine production [16].
Table 1 shows the primer sequences used and PCR conditions as described by a previous study with slight modifcations [24].Te amplifed products were then subjected to electrophoresis in a 1.5% agarose gel and visualized using a transilluminator.Evaluation of the correlation of carriage of the resistance genes bla TEM + bla CTX-M , bla TEM + bla OXA , bla TEM + bla SHV , bla CTX-M + bla OXA , and bla CTX-M + bla SHV with antibiotic resistance in P. aeruginosa isolates is shown in Table 6.Simultaneous carriage of two resistance genes bla TEM + bla CTX-M was found to pose a higher risk for P. aeruginosa resistance to IMP (OR � 2.511), AMX (OR � 2.401), FOX (OR � 2.521), and PPT (OR � 3.751); of two resistance genes bla TEM + bla SHV with higher risk for resistance to FOX (OR � 3.125) and bla CTX-M + bla SHV with higher risk for resistance to FOX (OR � 3.793) and PPT (OR � 2.120).International Journal of Microbiology

Discussion
Treatment of bacterial infections has been impaired due to the alarming rate of resistance that keeps on increasing with time especially those responsible for nosocomial and opportunistic infections [2].Te bacteria frequently responsible for nosocomial and opportunistic infections in Cameroon and the city of Douala are Gram-negative bacilli, including P. aeruginosa, which cause infection in immunocompromised patients [25].Te prevalence of P. aeruginosa is estimated to be between 7.1% and 7.3% among all nosocomial infections [26].P. aeruginosa is a particularly important pathogen in immunocompromised patients, particularly in patients with neutropenia.It is a critically important pathogen in patients with hematological malignancies, while also showing an increasing trend of MDR isolates in these patients [27,28].P. aeruginosa has now emerged as an ever-increasing problem due to its  Of the 507 participants, P. aeruginosa was isolated from 11.43% which is 77.58% from cancer patients and 22.42% from noncancer patients.On the other hand, more P. aeruginosa was isolated from cancer and noncancer patients belonging to the age groups of 40-49 years (31.11% and 23.07%) and 50-59 years (22 .22%and 23.07%), respectively.Tis result is higher than the 6.7% reported in an Indian study [29].Tis diference in prevalence could be due to diferences in sample size and immunity of participants.P. aeruginosa isolates exhibited signifcantly (p < 0.05) high resistance rates in cancer patients compared to noncancer patients: AMX (97.78% vs. 84.62%),IMP (82.22% versus 30.77%),CTX (97.78% versus 84.68).%),FOX (95.56% against 69.23%),CAZ (100% against 69.23%),PPT (100% against 69.23%),CBT (100% against 84.62%),CIP (93.33% versus 7.69%), and NAL (97.78% versus 7.69%).Contrary to the above, resistance rates of P. aeruginosa AMK (6.67% vs. 30.77%)and GEN (8.89% vs. 61.54%)were lower in participants with cancer than in those without cancer.Tese results show a marked increase in resistance of P. aeruginosa isolates in cancer patients compared to noncancer patients.Considering these results, the resistance of P. aeruginosa to beta-lactam antibiotics was very high.Garg et al. also found high levels of resistance to beta-lactam antibiotics in their study [30].Te increasing rate of Gram-negative bacilli towards cephalosporins could be explained by the fact that these antibiotics are commonly used to treat infections caused by the later inducing drug pressure.P. aeruginosa isolates were also highly resistant to quinolones and fuoroquinolones.Te very high rates of quinolone resistance observed in our work could be explained by the fact that exposure to fuoroquinolones is a risk factor for MDR Gram-negative bacilli infection in cancer patients [31].Frequent consumption of antibiotics without medical prescription in our community can lead to selective pressure of antibiotics on P. aeruginosa making this organism change its resistant mechanisms.We found an overall high rate of multidrug resistance among P. aeruginosa isolates and, importantly, a signifcant increase was observed in cancer patients compared to noncancer patients.Tese results are consistent with other reports on patients with hematological malignancies [32,33].Te emergence of resistance among P. aeruginosa isolates causing infection in neutropenic patients can be attributed to the administration of inadequate empiric antibiotic therapy, which severely impairs patient outcomes [33].Tis high rate of multiresistance as well as the high rate of resistance observed in cancer patients undergoing anticancer chemotherapy can be explained by the action of anticancer chemotherapy.Certain cytotoxic agents used in cancer treatment have antibacterial properties and have an impact on increasing bacterial mutation rates by activating the SOS response [34][35][36].Tis therefore contributes to the spread of antibiotic resistance mechanisms in bacterial populations in response to stress [37].Cancer patients are treated with substances that can modify bacterial DNA and increase bacterial mutation rates, leading to antibiotic resistance by accelerating the expansion of KPC-type β-lactamases (Klebsiella pneumoniae carbapenemase) [38][39][40][41].
P. aeruginosa is a pathogenic bacterium, causing nosocomial infections with intrinsic and acquired resistance mechanisms to a large group of antibiotics, including β-lactams.Cameroon is currently experiencing an increase in the number of bacterial infections associated with a broad  International Journal of Microbiology  International Journal of Microbiology spectrum of resistance to common antimicrobial agents.Te presence of ESBL-producing bacteria was reported this study.Molecular analysis revealed that bla CTX-M is the most prevalent (74.14%)ESBL gene followed by bla TEM , which was present in 72.41% of resistant strains.Te reason behind this increased frequency of occurrence of ESBL-producing organisms is likely due to the misuse and abuse use of antibiotics [42].It seems that the bla CTX-M type gene is the predominant genotype worldwide, particularly in Enterobacteriaceae [43].Few studies from other regions of the world have shown diferent prevalences of the bla CTX-M type gene among isolates, including 84.7% (Chile) and 98.8% (China) [44,45].We observed that the bla SHV and bla OXA genes were less common in our contexts, with a respective prevalence of 44.83% and 37.33%.A report from Hamad Medical Corporation, Qatar stated that the bla CTX-M type gene evolved through mutations in the bla TEM and bla SHV genes and is recently endemic [46].Tis phenomenon may pose serious risks to public health, as it would result in substantial limitations of therapeutic options.Tus, appropriate control measures, including establishing screening strategies to identify ESBLproducing bacteria, are necessary to prevent these strains from spreading.CTX-M was the most prevalent gene detected in ESBL-producing P. aeruginosa in the present study.Tis is consistent with a study conducted in the eastern region of Saudi Arabia, in which bla CTX-M type gene (97.4%) was more common than bla SHV (23.1%) and bla TEM (0.0%) [47].Likewise, studies in the eastern region have reported the predominance of the bla CTX-M type gene in Gramnegative bacteria producing ESBLs [48,49].As an illustration, bla CTX-M type gene was the most common type in the Asia-Pacifc region, followed by bla SHV and bla TEM [49].In Nigeria, the most frequent gene types among isolates from patients with surgical site infections were bla SHV , bla CTX-M , and bla OXA [50].In Burkina Faso, the most prevalent ESBL resistance genes were bla CTX-M (40.1%), bla TEM (26.2%), and bla SHV (5.9%) in Enterobacteriaceae [51].Tese results, together with the present fndings, revealed that the prevalence of ESBL gene types might vary from one geographical location to the other.
Although this study presents data on cancer patients with limited information on the resistance of P. aeruginosa to commonly used antibiotics, some limitations must be acknowledged.Te generalizability of the data could be compromised by sampling bias.Furthermore, the data should not be generalised to the whole country.Te study showed the prevalence of fecal carriage of ESBL-producing P. aeruginosa with a high rate of MDR in cancer patients however sequencing of the ESBLs studied in this work would be important in order to confrm TEM, SHV, or OXA ESBLs from other variants which are simple penicillinases.Despite the limitations mentioned, we remain convinced that this study provides essential information on the intestinal carriage of multidrug-resistant P. aeruginosa in cancer patients.Furthermore, plasmids are involved in gene transfer and carry additional antibiotic resistance genes as well as β-lactam resistance genes.Tese data therefore have important implications for the quality of patient care and infection control practices.

Conclusion
Te study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing P. aeruginosa with a high rate of MDR among cancer patients.bla CTX-M type gene was the most prevalent gene detected.Furthermore, the coexistence of two diferent ESBL genes was frequently detected in this bacterial pathogen.Regular monitoring and surveillance in the screening of ESBL-producing P. aeruginosa among cancer patients are needed to improve the management of patients.

Figure 2 :
Figure 2: Frequency of appearance of multidrug-resistant (MDR) Pseudomonas aeruginosa among cancer and noncancer patients.

Figure 4 :
Figure 4: Gel picture showing the amplifcation of the bla TEM gene fragment (445 bp).C−: negative control, C+: positive control, and M: molecular weight marker (100 bp ladder).Te lines 1 to 4 and 6 to 8 and 9 are positive isolates containing the bla TEM .

Figure 7 :
Figure 7: Efect of anticancer treatment on the ESBL genes of Pseudomonas aeruginosa. .

Table 1 :
Susceptibility of P. aeruginosa isolates was tested using nineteen diferent antibiotics during this study.Table2presents the susceptibility results of P. aeruginosa isolates to these antibiotics.P. aeruginosa isolates presented signifcantly (p < 0.05) high resistance rates in cancer patients compared to noncancer patients.OXA (37.93%, 22/58).More than half (74.14%, 43/58) of the isolates harbored more than one ESBL gene (Table3).Figures4-6present gel pictures of bla TEM , bla CTX-M , and bla SHV , respectively.Table4shows P. aeruginosa resistance genes in the diferent groups.Specifc PCR primers used in this study for the determination of antibiotic resistance genes.Figure 1: Distribution of Pseudomonas aeruginosa isolated according to diferent age groups.
Isolated according to Diferent Age Groups.Of 507 participants, P. aeruginosa was isolated in 58 (11.43%) participants, that is, 45 (77.58%) from cancer patients and 13 (22.42%)amongnoncancerpatients(Figure1).On the other hand, more P. aeruginosa was isolated in cancer and noncancer patients in the age groups of 25-34 years (31.11% and 23.07%) and 51-60 years (22.22% and 23.07%), respectively.3.2.Antibiotic Resistance Profle of Pseudomonas aeruginosaIsolates from Cancer and Noncancer Patients.3.3.Frequency of Multidrug-Resistant (MDR) Pseudomonas aeruginosa Isolates.Figure2shows the frequency of MDR in diferent isolates among cancer and noncancer participants.Te prevalence of MDR of P. aeruginosa was 96.55%.MDR P. aeruginosa isolates were signifcantly ((100% versus 84.62%; p < 0.05) elevated in patients sufering from cancer compared to noncancer patients.3.4.Efect of Anticancer Treatment on the Resistance Profle ofPseudomonas aeruginosa.Figure3shows the resistance profle of P. aeruginosa isolates based on anticancer therapy.Te fgure shows high resistance rates in cancer patients who underwent courses of chemotherapy compared to cancer patients without cancer treatment.3.5.Genotypic Detection of ESBL Carriage.Of the 58 isolates screened for ESBL production, 94.83% (55/58) harbored at least one ESBL gene.Te most prevalent gene detected in this study was the bla CTX-M type gene (74.14%,43/58)followed by bla TEM (72.41%, 42/58) and the least gene detected was bla 00%).Te results also show that the bla CTX-M type gene was signifcantly higher (p � 0.0001) in P. aeruginosa isolated from cancer patients (86.05%) compared to those isolated from noncancer patients (13.95%) and the bla SHV gene was signifcantly higher (p � 0.008) in P. aeruginosa isolated from cancer patients (88.46%) compared to those isolated from noncancer patients (11.54%).Figure7shows the International Journal of Microbiology

Table 2 :
Antibiotic resistance profle of Pseudomonas aeruginosa isolates from cancer and noncancer patients.Evaluation of the correlation of carriage of the resistance genes: bla TEM , bla OXA , bla CTX-M type gene, and Bla SHV with antibiotic resistance in P. aeruginosa isolates is shown in Table5.Tere was a signifcant correlation between carriage of the bla TEM gene in P. aeruginosa and resistance to antibiotics against CAZ (p � 0.032); carriage of the bla CTX-M type gene and resistance to PPT (p � 0.0262), CAZ (p � 0.0262) in P. aeruginosa.Carriage of bla OXA resistance genes was found to pose a higher risk for P. aeruginosa resistance to IMP (odds ratio (OR) � 2.541); bla SHV resistance genes with a higher risk for resistance to FOX (odds ratio (OR) �

Table 4 :
Frequency of ESBL genes among Pseudomonas aeruginosa isolates based on cancer status.

Table 5 :
Correlation of resistance genes and Pseudomonas aeruginosa resistance to antibiotics of the family.

Table 6 :
Correlation of two ESBL genes and aeruginosa resistance to β-lactam antibiotics.