Presence of the blaTEM Gene in Commensal Neisseria spp.: A Possible Cause for the Acquired Drug Resistance Among Pathogenic Respiratory Bacteria

Background The oral microbiome consists of various bacterial genera, with Neisseria spp. being a prominent part of this niche. While Neisseria gonorrhoeae and Neisseria meningitidis are human-restricted pathogens, non-pathogenic Neisseria species like Neisseria sicca, Neisseria perflava, etc., are primarily commensals that can also behave as opportunistic pathogens. With increasing penicillin resistance in commensal Neisseria, there is a concern that these bacteria might harbor resistance genes that can be transferred to other pathogens. This study aimed to characterize the blaTEM gene (encodes for the plasmid-mediated β-lactamase enzyme that hydrolyzes the β-lactam ring) of commensal Neisseria spp. isolated from respiratory samples. Methodology The research was conducted in the Department of Clinical Microbiology at Sri Ramachandra University, Chennai. The specimens used were sputum and throat swabs, which were subjected to a series of phenotypic methods and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) for speciation. The antibiogram was determined using the Kirby-Bauer disk diffusion method, and a PCR assay was utilized to identify the blaTEM gene responsible for β-lactamase production. Results Out of 274 processed samples, 65 unique commensal Neisseria spp. were identified. The study highlighted the presence of the blaTEM gene in 93.9% (61) of the isolates, which is responsible for β-lactamase production. All isolates exhibited resistance to penicillin. Most blaTEM-positive commensal Neisseria spp. were susceptible to cefuroxime (83.6%), ceftriaxone (85.2%), and cefotaxime (85.2%). The high prevalence of the blaTEM gene in commensal Neisseria is alarming. The gene, found on plasmids, could potentially transfer to other related species like Neisseria gonorrhoeae and Neisseria meningitidis, as well as other Gram-negative bacilli. Conclusion The presence of resistance genes in commensal bacteria is of concern, as they might be reservoirs for resistance transfer to pathogenic strains. The study emphasizes the importance of continuous monitoring and deeper investigations into commensal bacteria, emphasizing the need for a broader community screening approach to understand resistance mechanisms in the normal microbiome.


Introduction
The respiratory tract is divided into the upper and lower respiratory tracts.It is a well-known fact that upper respiratory is pooled with commensal microbial flora like Group A, C, G Streptococci, Streptococcus pneumoniae, other α-haemolytic Streptococci, non-typeable Haemophilus influenza, Moraxella catarrhalis, Neisseria meningitidis, commensal Neisseria spp., Staphylococcus aureus, Enterococcus species, Enterobacteriaceae, some coliforms, and non-fermenters [1,2].Potentially pathogenic organisms such as Haemophilus, Mycoplasma, and Pneumococci may also be found in the pharynx, along with Gram-negative anaerobes like Prevotella, Porphyromonas, Fusobacterium, and Veillonella.Grampositive anaerobes like Finegoldia, Parvimonas, Bifidobacterium, Eubacterium, etc.Even though colonization of pathogens is prevented by a few natural protective mechanisms like the flow of saliva, secretary antibodies (IgA), lysozyme that participate in the destruction of bacterial cells, and the presence of normal flora (protective microorganisms), which produce substances that hinder successful invasion by harmful organisms and also act as physical barriers.The upper respiratory tract is often the site of initial colonization for the pathogens (Neisseria meningitidis, Corynebacterium diphtheriae, Bordetella pertussis, and many others) and is considered the first region of attack for these organisms.In contrast, the lower respiratory tract (small bronchi and alveoli) is usually sterile.If bacteria do reach these regions, they encounter host defense mechanisms, such as alveolar macrophages, that are not present in the pharynx [3,1].
Members of the genus Neisseria are gram-negative diplococci, which are usually the normal residents of the mucous membranes of mammals and reptilians; some species are primary pathogens for humans (Neisseria gonorrhoeae and Neisseria meningitidis).The commensal Neisseria are largely confined to the upper respiratory tract of humans.All organisms belonging to the Neisseriaceae are aerobic.They are oxidase and catalase-positive (except Neisseria elongata, which is catalase-negative).They colonize mucosal surfaces, generally without causing overt pathology, but may also cause infectious diseases when the host becomes vulnerable [4].

Drug resistance
Like other organisms, commensal Neisseria can also naturally transfer resistance genes to closely related species.They might constitute a DNA source for the emergence of antibiotic resistance in meningococci, as evident by the recent appearance of moderate resistance to penicillin in pathogenic Neisseria.The penicillin non-susceptibility is due to altered PBP2 (encoded by the penA gene) or, in other cases, to the production of a plasmid-encoded β-lactamase, causing high-level resistance [5].Multi-resistant plasmids containing the TEM-1 β-lactamase have been isolated from oral commensal Neisseria spp. as well [6].The spread of a potent ESBL from the commensal Neisseria spp. to the Neisseria gonorrhoeae population results in the degradation of ceftriaxone and the development of drug resistance in Neisseria gonorrhoeae [7].
In this pursuit, the current study was undertaken to look for the presence of the drug resistance gene among the commensal Neisseria spp.isolated from respiratory samples using phenotypic as well as molecular methods.

Materials And Methods
A cross-sectional study was conducted in the Department of Clinical Microbiology, Sri Ramachandra Laboratory Services (SRLS), Sri Ramachandra Institute of Higher Education and Research (SRIHER), Sri Ramachandra University, Chennai, after obtaining institutional ethics committee approval (CSP-MED/20/SEP/61/73).All the upper respiratory samples, consisting of sputum and throat swab clinical specimens, were subjected to Gram staining for the presence of Gram-negative diplococci and cultured on blood agar and chocolate agar plates.The plates were incubated overnight at 37°C.Neisseria spp. was phenotypically identified based on the growth and colony morphology on blood agar and chocolate agar plates.Biochemically, oxidase and catalase tests were used for the identification.Antibiotic susceptibility to various classes of antibiotics was determined using commercially procured antibiotic discs by the disc diffusion method in accordance with CLSI 2021 M-100 guidelines [8].The antibiotics tested were penicillin (10 units), cefuroxime (30µg), ceftriaxone (15µg), tetracycline (30µg), cefotaxime (30µg), azithromycin (30µg) and ciprofloxacin (30µg).As per the CLSI 2021 M-100 guidelines for Neisseria gonorrhoeae, the results were interpreted as susceptible, intermediate, and resistant [8].Polymerase chain reaction (PCR) for detection of the blaTEM gene was done using PureFast® Bacterial DNA minispin using primers obtained from HELINI Biomolecules.The primer used was TEM-F: TTTCGTGTCGCCCTTATTCC and TEM-R: ATCGTTGTCAGAAGTAAGTTGG at 260 bp.

Results
Of the 274 respiratory samples processed during the study period, 65 showed growth of commensal Neisseria spp.(Figures 1-3).Polymerase chain reaction (PCR): PCR screening for blaTEM genes revealed 61 (93.9%; n=65) isolates had blaTEM genes in our study (Figure 4).The remaining four (6.1%) did not harbor the blaTEM gene.The antibiotic susceptibility pattern in comparison with the presence or absence of the blaTEM gene is shown in Table 3. samples was 28%, which was in concordance with our study, which had 22.6% of Neisseria spp.from sputum samples and 30.5% from throat swabs [9].Based on our study, the throat swab seems to be a better specimen of choice for the isolation of commensal Neisseria.Mechergui et al. in 2014 [10] reported 100% correct identification of commensal Neisseria spp.by MALDI-TOF.In our study, we speciated commensal Neisseria both by conventional phenotypic methods and by mass spectroscopy and were able to identify five different species, namely N. flava/subflava/perflava complex, N. flavescens, N. mucosa, N. sicca, and N. elongata, both of which were comparable.

Antibiotics
Various authors have reported different quantums of beta-lactamase production due to the presence of the blaTEM gene in commensal Neisseria, viz., Obi M.C. et al. [9] in 1990 at Lagos, Nigeria, showed 33% by the phenotypic method, and Mechergui et al. [5] of Tunisia in 2011 reported 9% by the genotypic method.In our study, 93.9% of isolates showed the presence of the blaTEM gene by PCR, a representative of beta-lactamase production, indicating that the increased usage of beta-lactam antibiotics of late may be the reason.However, about 86% of our isolates were susceptible to second and third generation cephalosporins, indicating that the blaTEM gene present in the isolates might not have been expressed and may be expressed in the presence of a substrate, i.e., cephalosporins.We have not estimated the detection of penA genes, which are responsible for penicillin resistance.
A three-year study done in 2003 by Mechergui et al. [5] in Tunisia showed penicillin resistance of 34%, but our study shows penicillin resistance of almost 100%.Even a study done in Japan by R. Furuya et al. [11] between 2005 and 2006 showed 8.8% of penicillin resistance and 88.9% of intermediate resistance to penicillin by N. subflava.However, our N. subflava isolates showed 90.3% penicillin resistance, and 9.7% showed intermediate resistance.A Spanish study done by L. Arreaza et al. [12] in 2002 found that commensal Neisseria had a 100% intermediate susceptibility to penicillin for Neisseria lactamica.In 2015, Mechergui et al. [5] did antimicrobial susceptibility testing for commensal Neisseria spp. in Tunisia, which reported 87% resistance to penicillin and the rest of the isolates to be intermediately susceptible to penicillin, which correlates with our findings.
One of the studies done in New York, USA, by Michael A. Fiore et al. [13] in 2020 showed penicillin resistance of 19.2%, followed by cefixime resistance of 11.9% and ceftriaxone resistance of 7.6%.In our study, we found 13.9% resistance to 3rd-generation cephalosporins and almost 100% resistance to penicillin.The same study showed ciprofloxacin resistance of 7% and tetracycline resistance of 14%, respectively.In our study, we had ciprofloxacin resistance of 18.5% and tetracycline resistance of 20%, respectively, which contrasts with the Japanese study done by R Furuya et al. [11], which showed 44.4% intermediate resistance and 31.1% resistance to ciprofloxacin and 60% intermediately resistant and 28.9% resistant to tetracycline in N. subflava isolates.In our study, the N. subflava isolates showed 16.13% resistance and 6.45% intermediate resistance to ciprofloxacin.A study done by Michael A. Fiore et al. [13] in 2020 in New York, USA, showed ciprofloxacin resistance to be 15.4%, which is in concordance with our study.The same study had macrolide resistance of about 42.3%, but our study showed 35% resistance to erythromycin; however, we have also tested for azithromycin susceptibility as per CLSI 2021, which showed a resistance of 7.7%.

Drug resistance gene
Our study had 93.9% blaTEM by conventional PCR.However, the Tunisian study showed the presence of blaTEM in 9% of their isolates [5].In this study, the identification of the TEM-1 gene in 93.9% of commensal Neisseria is alarming, as it is well proven that the resistance gene can horizontally transfer to pathogenic Neisseria (Neisseria gonnorhoea and Neisseria meningitidis), thus making the previously susceptible pathogenic strains into resistant strains.The possible reason for acquiring resistance could be because of the irrational and indiscriminate use of antibiotics in poultry feed [14].
The limitations of the study are screening of 274 samples had only isolation of 65 commensal Neisseria spp.Inclusion of a greater number of commensal Neisseria spp.would have been better for more information, and also the clonal relatedness of blaTEM gene between the commensal Neisseria spp.and the pathogens is not performed.

Conclusions
The possibility of the blaTEM gene, which is present in the plasmid of commensal Neisseria, getting transferred to other closely related species like Neisseria gonnorhoea and Nesseria meningitidis.This could be one of the prime reasons for acquiring drug resistance among the pathogenic bacteria, especially in the hospital environment.A few SNPs (single nucleotide polymorphisms) in the beta-lactamase TEM gene will convert them into extended-spectrum beta-lactamases and give them resistance to other classes of antibiotics as well.The source of blaTEM in commensal Neisseria is yet unknown.A community screening for the presence of the blaTEM gene may shed light on this state.According to our study, a throat swab seems to be a good specimen for screening blaTEM in commensal bacteria.Since most of the studies are being done on pathogenic Neisseria, a large multi-center study in commensal Neisseria may give us hints for explaining the source of resistance mechanisms present in normal microbiomes transferring to probable pathogenic organisms.

FIGURE 3 :
FIGURE 3: Growth of Nesseria on chocolate agar shows yellow-colored colonies

FIGURE 4 :
FIGURE 4: Gel electrophoresis of PCR for detecting the blaTEM gene (the band at 260 bp represents the presence of the blaTEM gene) Lane 1: 1000-bp ladder, Lane 2: Negative control, Lane 3: Positive control, Lane 4: Negative for blaTEM gene, Lane 5: Positive for the blaTEM gene, Lane 6: Negative for the blaTEM gene, Lane 7: Negative for the blaTEM gene

TABLE 1 : Age-wise distribution of the Neisseria spp. isolated in the study group
Neisseria elongata, which was catalase negative and appeared in coccobacillary form in the gram smear.Antibiotic susceptibility testing revealed that except for the four isolates that showed intermediate susceptibility to penicillin, all others were resistant to penicillin.N. elongata was found to be susceptible to all tested antibiotics except penicillin.Azithromycin had a higher susceptibility rate of 92.3%, followed by ceftriaxone and cefotaxime at 86.1% each.The details of the antibiotic susceptibility report for the Neisseria test isolates are shown in Table2.

TABLE 3 : Comparison of antibiotic susceptibility among the blaTEM gene-positive and negative commensal Neisseria isolates Discussion Of
the total number of 274 samples, 65 (23.7%)grew commensal Neisseria spp.The colonization of Neisseria spp. was less in both age extremes (3.1% in both the less than 10 years and more than 71 years age groups).In our study, colonization by commensal Neisseria was observed after five years of age.The lesser number of colonizations in the older age group may be indicative of a poor oral milieu for the survival of commensal Neisseria, which needs further evaluation with a larger sample size.Early phenotypic studies done by Obi M.C. et al. in 1990 at Lagos, Nigeria, showed that the carriage of commensal Neisseria spp. in sputum

Table 4
shows the comparison of the commensal Neisseria spp.antibiotic susceptibility among the various studies conducted.