Resistance of Vibrio cholera to antibiotics that inhibit cell wall synthesis: A systematic review and meta-analysis

Objective: Cholera is a challenging ancient disease caused by Vibrio cholera (V. cholera). Antibiotics that prevent cell wall synthesis are among the first known antibiotic groups. Due to its high consumption, V. cholera has developed resistance to the majority of antibiotics in this class. Resistance to recommended antibiotics for the treatment of V. cholera has also increased. In light of the decrease in consumption of certain antibiotics in this group that inhibit cell wall synthesis and the implementation of new antibiotics, it is necessary to determine the antibiotic resistance pattern of V. cholera and to employ the most effective treatment antibiotic. Method: An comprehensive systematic search for relevant articles was conducted in PubMed, Web of Science, Scopus, and EMBASE through October 2020. Stata version 17.1 utilized the Metaprop package to execute a Freeman-Tukey double arcsine transformation in order to estimate weighted pooled proportions. Results: A total of 131 articles were included in the meta-analysis. Ampicillin was the most investigated antibiotic. The prevalence of antibiotic resistance was in order aztreonam (0%), cefepime (0%), imipenem (0%), meropenem (3%), fosfomycin (4%), ceftazidime (5%), cephalothin (7%), augmentin (8%), cefalexin (8%), ceftriaxone (9%), cefuroxime (9%), cefotaxime (15%), cefixime (37%), amoxicillin (42%), penicillin (44%), ampicillin (48%), cefoxitin (50%), cefamandole (56%), polymyxin-B (77%), carbenicillin (95%) respectively. Discussion: Aztreonam, cefepime, and imipenem are the most efficient V. cholera cell wall synthesis inhibitors. There has been an increase in resistance to antibiotics such as cephalothin, ceftriaxone, amoxicillin, and meropenem. Over the years, resistance to penicillin, ceftazidime, and cefotaxime, has decreased.


Introduction
Koch isolated and described the Gram-negative bacillus Vibrio cholera (V. cholera) in the late nineteenth century (Dengo-Baloi et al., 2017). As a foodborne and waterborne pathogen, V. cholera can cause an acute intestinal infection as severe watery diarrhea in humans (Mukhopadhyay et al., 1998;Yuan et al., 2022). Virulence factors include toxin-related fimbria and cholera toxins (Ngandjio et al., 2009;Awuor et al., 2020a). In areas with poor sanitation and no clean water, V. cholera can be endemic, epidemic, or pandemic (Mukhopadhyay et al., 1998). This bacterium causes 2.9 million cholera cases and 95,000 deaths annually (Onohuean et al., 2022). According to the O antigen, V. cholera species are divided into 206 serotypes. Although the O1 and O139 serotypes are linked to epidemic cholera and non-agglutinating V. cholera (NOVC), which are negative to the O1 and O139 antigens, they cause infrequent severe illnesses (Liu et al., 2022;Onohuean et al., 2022). Seventh cholera pandemics linked to O1 and O139 V. cholera serotypes. The disease is a major issue in Asia, Africa, and Latin America (Abera et al., 2010;Alam et al., 2012). V. cholera infections can be mild, moderate, or severe in endemic areas (rapidly deadly diarrhea) (Araj et al., 1994;Anand et al., 1996).
Early treatment with an oral rehydration salts (ORS) solution, including glucose, potassium chloride, sodium chloride, and trisodium citrate, are critical for cholera patients with moderate watery diarrhea (Araj et al., 1994). Severe cholera dehydration requires intravenous rehydration and appropriate antibiotics to shorten the disease's period (Araj et al., 1994;Baddam et al., 2020).
Although antibiotic susceptibility testing (AST) of V. cholera was not suggested in the past due to the low resistance of V. cholera to common antibiotics (Bag et al., 1998;Baddam et al., 2020), the development of resistance to tetracycline, a common antibiotic used in the treatment of V. cholera infection, is becoming increasingly widespread throughout the world (Bakhshi and Pourshafie, 2009). These resistant strains have been responsible for severe epidemics in Latin America, Tanzania, Bangladesh, and Zaire (Bag et al., 1998). However, various isolates of V. cholera resistant to antibiotics have been reported worldwide. Antibiotics that target the cell wall were once the first-line treatment for infections, but their use has decreased as bacterial resistance to these antibiotics has increased over time. Globally, the prevalence of V. cholera resistance to cell wall-active antibiotics has not been thoroughly studied. Replacing less effective antibiotics is necessary for more effective treatment, which has already increased the prevalence of resistance.
The tetracycline antibiotic class has long been the most effective for treating cholera. Non-etheless, earlier research revealed a global increase in V. cholera strains resistant to tetracycline (Dengo-Baloi et al., 2017). Additionally, studies have demonstrated that fluoroquinolone resistance in V. cholera strains began to increase in July 1996 (Mukhopadhyay et al., 1998;Yuan et al., 2022). A previously published study suggested erythromycin as a tetracycline alternative in small children and pregnant women. Furazolidone and nalidixic acid have traditionally been used as cholera treatments.
Nevertheless, due to the high level of resistance found in V. cholera isolates, these antibiotics are currently less effective (Ngandjio et al., 2009;Awuor et al., 2020b). Due to the lack of meta-analyses concerning antibiotics resistant to the V. cholera cell wall, we decided to explore newer antibiotics in terms of the prevalence of resistance as well as the global resistance pattern and the resistance trend over time in all V. cholera serotypes. This study's outcomes can potentially improve the global antimicrobial resistance situation significantly.

Search strategy
The comprehensive systematic search of relevant articles through four electronic databases, including PubMed, Web of Science, Scopus, and EMBASE, with two researchers independently until October 2020. The search was performed using "V. cholera" and "Antibiotic resistance" related keywords. Obtained articles have been merged in EndNote X20 (Thomson Reuters, NY, United States), and duplicates were removed. The search syntax is available in Supplementary Material.

Selection criteria and data extraction
The screening and selection of articles procedures were performed in Rayyan online software. Two authors (A. Sh and O. Sh) independently reviewed all records' titles, abstracts, and full texts. They removed irrelevant articles, and the third author (M. Sh) solved disagreements. The exclusion criteria were as follows: review articles, case reports, congress abstracts, studies with ambiguous results, not the English language, sample size of fewer than three isolates, duplicate publications, and studies of antimicrobial resistance of other than V. cholera species.
The extracted information from each included study was: first author, year of publication, country, sample source (clinical or environmental isolates), serogroups, the total number of isolates (sample size), AST method, and the number of resistant isolates for each antibiotic.

Quality assessment
The quality assessment of included studies was performed by two reviewers (R. Sa, and O. Sh) independently using an adapted version of the Newcastle-Ottawa assessment scale adapted for crosssectional studies. Each study received a score ranging from 0 to 8 (5 points or higher: high quality, three or 4: medium quality, 2 points or lower: low quality).

Statistical analysis
Due to the high number of zero prevalence in antibiotic resistance reports, the Freeman Tukey double arcsine conversion was conducted on the data using the metaprop command to estimate the weighted pooled proportion of resistance (WPR) in STATA software (version. 17.1), which range is from 0.00 to 1.00. A random-effects model was used for pooling effect size. As a measure of heterogeneity, the tau-squared and I 2 were considered. The Egger regression test was used to determine the effect of small studies or Frontiers in Pharmacology frontiersin.org 02 publication bias. Subgroup analyses were conducted using the following variables to find the sources of variation: country, continent, country development status (World Economic Situation and Prospects, classification), publication year group (1970-2000, 2001-2010, 2011-2020), source of V. cholera isolation, AST method (Disc and Gradient methods), interpretation guideline (CLSI, Non-CLSI), and serogroups (O1/ O139, Non-O1/O139).

FIGURE 1
The study Prisma flow diagram.
The WPR of V. cholera to carbenicillin was 0.95 (95% CI [0.65, 1.00]), and the heterogeneity was significant (I 2 = 85.02, p < 0.01). Malaysia and India have the highest WPRs (WPR; 1.00, 1.00, respectively). All of the studies have been in developed countries. Clinical isolates had a substantially higher WPR than environmental isolates (WPR; 1.00, 0.54, respectively). The WPR in O1/ O139 serogroup was higher than the non-O1/O139 serogroup (WPR; 1.00 and 0.54, respectively). All studies have used CLSI as an AST guideline. The heterogeneity of subgrouping based on the AST methods was insignificant (p = 0.52).
The heterogeneity between countries and sources of isolates was significant (p < 0.05). The heterogeneity between the AST method and serogroups was insignificant (p = 0.072). All studies used CLSI guidelines.

Resistance to carbapenems
The V. cholera WPR to imipenem is 0.00 (95% CI [0.00-0.02]), and heterogeneity was significant (I 2 = 79.95, p < 0.01). India and Germany had the highest WPR (0.05 and 0.02, respectively). CLSI was utilized as a guideline in all studies. The heterogeneity of subgrouping based on the continents, AST method, AST guideline, isolated sources, and serogroups were insignificant (p > 0.05).

Resistance to polymyxin B
The V. cholera WPR to polymyxin B is 0.77 (95% CI [0.54, 0.94]), and the heterogeneity was significant (I 2 = 98.24, p < 0.01). The countries with the highest WPR were India and Iran (WPR; 0.86, 0.65, respectively). The WPR of the CLSI subgroup was higher than the non-CLSI group (WPR; 0.78, 0.16, respectively). The heterogeneity of subgrouping based on the serogroups and isolate sources was insignificant (p > 0.05).

Discussion
Cholera is a historical, unresolved problem and a severe healththreatening infection, particularly in developing countries. This  (19). Due to V. cholera's low resistance, the AST of these microorganisms was avoided in the past. (14, 15). However, the emergence of β-lactam-resistant species has had severe repercussions on managing infectious diseases globally (16). Tetracyclines have long been the antibiotic for treating severe cholera (15). Conversely, tetracycline-resistant V. cholera strains are spreading internationally (17). Severe epidemics in Latin America, Tanzania, Bangladesh, and Zaire have been linked to tetracycline-resistant strains (15). A meta-analysis with 52 studies has estimated the tetracycline and doxycycline resistance rate of V. cholera serotype O1 over 50% and 28%, respectively (20). This escalating resistance rate underscores the importance of regulating antibiotic prescriptions and discovering effective alternatives.
The current study investigated V. cholera cell wall-targeting antibiotic resistance patterns. This study revealed that carbenicillin (95%), and polymyxin B (77%) have the highest resistance rates. The ampicillin, penicillin, and amoxicillin resistance rates were 48%, 44%, and 42%, respectively. Adding clavulanate acid to amoxicillin reduced the proportion of resistant bacteria to 8%, which was more effective than amoxicillin alone.
The rates of resistance to the first generation of cephalosporins, such as cefalexin and cephalothin, were 8% and 7%, respectively. The percentages of resistance to the second generation of cephalosporins, including cefamandole, cefoxitin, and cefuroxime, were 50%, 56%, and 9%, respectively. Compared to the first generation of cephalosporins, V. cholera was more resistant to the second generation. Cefixime (37%), cefotaxime (15%), ceftazidime (5%), and ceftriaxone (9%) As members of the third generation of cephalosporins are more effective than second-generation cephalosporins. Cefepime is the only member of the fourth generation of cephalosporins studied in four articles evaluating 609 V. cholera isolates, and none of them were resistant to it.
The rate of aztreonam resistance was 0%, while the rate of fosfomycin resistance was 4%. Cefepime, aztreonam, and imipenem were the most efficient antibiotics against V. cholera. Non-etheless, the heterogeneity between reported imipenem resistance rates was substantial, and it appears necessary to perform AST before prescribing antibiotics to ensure the isolate's susceptibility.
Considering sulfonamides are commonly used to treat HIV, TB, malaria, pneumonia, and febrile illness, Onohuean et al. (2022) estimated the prevalence of quinolone, tetracycline, and sulfonamide resistance genes to be 32.97 percent ). This percentage is lower than that of polymyxin and higher than that of cephalosporins. An additional study was analyzed from a total of 139 articles involving 24,062 isolates of V. cholera O1/O139. Asia was the location of origin for 102 out of the total research. The WPR was calculated as follows: azithromycin had a 1%, erythromycin 36%, ciprofloxacin 3%, cotrimoxazole 79%, doxycycline 7%, and tetracycline had a success rate of 20% WPR. Between the years 1970- 2000 2001-2010 2011-2020 Heterogeneity between groups Frontiers in Pharmacology frontiersin.org 08 1980 and 2020, there was a growth in drug resistance to cotrimoxazole, ciprofloxacin, and tetracycline (Liu et al., 2022).
According to the findings of Xin-hui Yuan's research, there has been an increase in drug resistance in recent years, particularly to nalidixic acid, cotrimoxazole, furazolidone, and tetracycline. Between 2000 and 2020, however, resistance to antibiotics such as amoxicillin, ciprofloxacin, erythromycin, chloramphenicol, ampicillin, streptomycin, and ceftriaxone decreased. The frequency of doxycycline and ciprofloxacin resistance in V. cholera O1/O139 isolates significantly reduced from 2011 to 2020 compared to the frequency of these resistances from 2001 to 2010 (p < 0.05) (Yuan et al., 2022).
Based on subgroup analysis of continents, Africa and Asia had the highest proportion of resistant individuals. Patterns of resistance vary between developing and developed nations, and the resistance rate in developing nations was significantly higher than in developed nations. Even though developed countries had more resistant bacteria to amoxicillin, aztreonam, imipenem, and fosfomycin, developing countries had more resistant bacteria to augmentin, penicillin, ampicillin, cefotaxime, ceftazidime, and ceftriaxone.
Antibiotics to which V. cholera is highly resistant in developed nations have not been studied in developing countries. Cefixime, cefoxitin, cefuroxime, cefamandole, polymyxin B, cephalothin, and carbenicillin appear to have been taken off the list of V. cholera treatments. There has been an increase in resistance to antibiotics such as cephalothin, ceftriaxone, amoxicillin, and meropenem. Over the years, resistance to penicillin, ceftazidime, and cefotaxime has decreased. Because cholera was historically treated with numerous antibiotics, resistance to these antibiotics grew over time. For instance, penicillin resistance reached 98% during a specific time frame. After this increase in V. cholera infection treatment, it appears that this antibiotic was no longer considered. In recent years, the resistance to this antibiotic has decreased significantly, reaching 19% due to the lack of prescribing and use.

Conclusion
Our findings indicate the importance of prescribing antibiotics accurately to control and prevent V. cholera antibiotic resistance. Following the disappointing emergence of antibiotic resistance to certain antibiotics, such as penicillin, these antibiotics were utilized less frequently, which resulted in a decrease in antibiotic resistance to these antibiotics in general. This event increases optimism that using old antibiotics will be effective if antibiotic use is controlled. In that order, the antibiotics with the least resistance to V. cholera were cefepime, aztreonam, imipenem, and meropenem. Due to the vastly Frontiers in Pharmacology frontiersin.org 09 different patterns of antibiotic resistance of V. cholera to these antibiotics in various geographic locations, it appears necessary to investigate the antibiotic resistance of the isolates.

Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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