This work was taken up to establish the possible spread of antibiotic resistance through the bacteria isolated from water samples including influents, effluents, surface and ground water samples collected in and around pharmaceutical industries of south India. In this study, 48 bacterial isolates from different water samples, were used that includes Gram negative bacilli, Klebsiella pneumoniae (7), Pseudomonas aeruginosa (9), Enterobacter aerogenes (6), Acinetobacter sp. (2), Aeromonas punctata (2), Ralstonia picketti (1), Stenotrophomonas maltophillia (2), Citrobacter freundii (2) and Gram-positive cocci, Staphylococcus aureus (16), Corynebacterium sp, (1). In terms of percent prevalence among the isolates, Staphylococcus aureus − 28%, Pseudomonas aeruginosa − 22%, Klebsiella pneumoniae − 19%, Enterobacter aerogenes − 12%, Acinetobacter sp. − 5%, Citrobacter freundii − 4%, Aeromonas punctata − 3%, Stenotrophomonas maltophillia − 3%, Corynebacterium sp. − 2%, Ralstonia picketti − 2%, were noticed. Though this study was focused on pharmaceutical industry wastewater analysis of bacterial isolates. In the present work, comprehensive study has been performed for the first time to assess the status of phenotypic and genotypic profile of AMR along with antibiotic residue analysis in pharmaceutical industry waste water, surrounding surface and ground water. A recent study had analyzed influent waste water samples from municipal of Helsinki in Finland. Upon characterizing the bacterial isolates by phenotypic and genotypic methods, they found predominantly beta lactamase resistance mediating ESBL genes.
The influent waste water samples had a total of 30 isolates (Gram positive – 11 Nos. and Gram negative – 19 Nos.) among 48 isolates used in this study but only one effluent sample had one isolate of Gram-negative type which clearly indicates that the waste water treatment plant is almost working effectively. At the same time, it is also a major concern with regard to 17 isolates obtained from environmental samples like surface and ground water samples, giving a caution about other source of contamination and spread of resistant bacteria which may lead to a major public health issue. This address the need to explore the source and spread of antibiotic resistant bacteria in the environment. One study has been carried out by28 in Mysuru, India. It established the environmental contamination of four medicinal compounds including ceftriaxone in municipal wastewater treatment plant effluent. In another study conducted in Nigeria, it was revealed that the release of wastewater from slaughterhouses directly into water sources without prior treatment, has led to significant health hazards due to multidrug resistant bacterial population contaminating the environment29. A high resistance to these β-lactam antibiotics is expected, given that ampicillin is among the frequently prescribed antibiotics for treating infections in both humans and animals. Moreover, resistance to penicillin in Staphylococcus aureus has been documented since the 1960s. Additionally, ampicillin-resistant isolates including MRSA may exhibit cross-selection for resistance to other beta-lactams30.
Another supporting evidence of environmental contamination was evidenced by phenotypic analysis of bacterial isolates from water samples collected after treatment in a study conducted in Mafikeng in the North West Province of South Africa. This study identified P. aeruginosa in both drinking and surface waters. However, in contrast to our results, a study in Alice, Eastern Cape, South Africa, analyzing wastewater samples, reported a lower occurrence rate of 11.1%. This difference is likely attributed to variations in water purification treatment processes, or it can be inferred that wastewater treatment plants (WWTP) may not completely eradicate bacteria, especially multidrug-resistant (MDR) strains. These resilient organisms might withstand treatment processes, contributing to the transmission and spread of antimicrobial resistance 31 .But in this study, only one Enterobacter aerogenes (18: KA/E/2021) isolate was obtained exhibiting resistance to all antibiotics tested in this study indicating the effectiveness of the wastewater treatment process.
The emergence of bacterial resistance to most frequently used antibiotics/drugs is of immense medical significance due to the public health hazards. Multiple investigations in polluted and non-polluted areas have confirmed the occurrence of antibiotic resistant microorganisms 5. Genetic flexibilities have enabled them to survive in a wide range of environments due to their capacity to obtain and transfer drug resistance. Aquatic environments promote gene transfer amongst strains of bacteria 32. In this study also, ARGs transfer through water sources in the environment was established even though the source of antibiotic resistant bacteria development is not known. This needs to be explored with epidemiologic and typing methods for clonal relatedness.
In this study, forty-eight bacterial isolates of Gram positive and Gram-negative types were tested for their antibiotic susceptibility and resistance against sixteen antibiotics (Table 6).
All the Gram-positive isolates tested had phenotypic resistance against cephalosporins, lincosamide, polypeptide antibiotics, aminoglycoside, and penicillin and macrolide group of antibiotics. Glycopeptide and oxazolidinone group of antibiotics were the only one to which all strains were susceptible. Among the Gram-negatives, 24 out of 31 isolates were resistant due to ESBL. Fluroquinolones and aminoglycoside resistance
mediating genes occurred in 7 isolates. Whereas earlier reports had shown higher prevalence of multidrug resistant bacteria because the samples were sourced from common treatment plant compared with this study which focused on few pharma industries only. Acinetobacter sp., Klebsiella pneumoniae, Proteus mirabilis, Enterobacter sp., and Bacillus sp. estimated for the bulk of the 254 bacterial strains that were isolated; 218 (85.8%) of these isolates were multidrug resistant 33.The resistance patterns of the isolates indicated a resistance rate of 63.9% to second-generation cephalosporins (ceftazidime) and 55.6% to third-generation cephalosporins (cefepime), while displaying lower resistance to aminoglycosides. The resistance pattern observed for cephalosporins in the current study aligns with the findings of a previous report but is lower when compared to another study that reported 100% resistance rate. However, in yet another study, it was documented lower resistance rates of 6.9% and 17% to ceftazidime and cefepime, respectively34.
With regard to genotypic resistance pattern, among the 17 Gram-positive isolates, only 3 isolates (two influent and one ground water samples) had ARGs for aminoglycoside, fluroquinolones and beta lactam groups. Though the above group’s antibiotic resistance was phenotypically positive and the genotypic expression of those groups was not evident. They possible harvarded other resistance mediator. Among the Gram-negative isolates, 10
out of 12 ARGs were present in all 31 isolates and as expectedly, ARG viz. aac (3)-XI were not present in any of the isolates. This indicates that the beta lactam, fluroquinolones, quinolones and aminoglycosides groups ARGs were prevalent among Gram-negative bacteria. Normally, pharmaceutical industries liquid waste tends to discharge huge varieties of ARGs compared to common sewage and this was proved earlier by35. where they have observed the diversity of ARGs in pharmaceutical wastewater treatment plant aerobic sludge (153 subtypes) was higher than that in sewage treatment plant aerobic sludge (118 subtypes). A total of 12 antibiotics were checked for their effectiveness against the isolates. From this study along with earlier reports, it has been well established that the waste water treatment is an essential process to prevent or reduce the antimicrobial resistance development and spread in the environment 36–38 .These reports have shown higher level of phenotypic and genotypic resistance development and spread in the absence of wastewater treatment plant in the environment.
The correlation matrix analysis among Gram positive isolates revealed that in the presence of beta lactam groups resistance, aminoglycoside and fluroquinolones resistance were not present. Similarly, macrolides resistance never co-existed with aminoglycosides and fluroquinolones. But, all beta lactam group antibiotics resistance was present along with macrolides resistance. Similarly, in Gram-negative isolates, fluroquinolone ARGs never co-existed with beta lactam group antibiotics and beta lactam ORGs with aminoglycoside and other quinolone groups. But, in this study, a strong correlation was shown that some beta lactam and fluroquinoloe antibiotics resistance were co-existed; there were also evidences for the co-existence of fluroquinolone, beta lactam group ARGs with beta lactams, fluroquinolones and aminoglycoside antibiotic groups39,40. Previous study demonstrated that genotypic factors play a significant role in influencing phenotypic antibiotic resistance41.
This study had disclosed considerable prevalence of resistance toward diverse group of antibiotics, particularly aminoglycosides, macrolides, fluroquinolones, cephalosporin and β-Lactamase, which was established by the presence of blaOXA−58, blaOXA−22, qnrA, qnrB, aac(6)-Ib-cr, aac (3)-XI, mec A, qepA, aadB, blaVIM, blaOXA−48 and blaNDM antibiotic resistant genes, respectively. The phenotypic resistance profile of bacterial isolates was confirmed by the genotypic testing. The variations encountered between phenotypic and genotypic antibiotic resistance is attributed to the resistance expression level of diverse genes in the bacteria.