This study found that about one-third of women (35%) with PROM seeking care at MRRH had cervical amniotic bacterial colonization. The most common bacteria isolate was Klebsiella pneumoniae followed by Staphylococcus aureus. There was good sensitivity to quinolones and cephalosporins and marked resistance to penicillins. Cervical amniotic bacterial colonization was associated with being a prime gravida, being obese and being referred in. Overall these findings highlight the need to periodically review and update guidelines for the prophylactic use of antibiotics in PROM management; revising treatment protocols and considering alternative antibiotics based on local resistance patterns could improve patient outcomes and prevent complications associated with ineffective antibiotic therapy.
The prevalence of cervical amniotic bacterial colonization of 35% reported in the current study is consistent with findings from studies conducted at Mulago Hospital, Uganda (30% in 2017), and Wayne State University, USA (41% in 2015) [6] [5]. However, a study at Stanford University, USA in 2010 reported a higher prevalence of 50% [18]. The similarity of our findings with those at Wayne University could be attributed to the use of universal primers for PCR, which allows for the detection of a broad range of bacteria. In contrast, the higher prevalence at Stanford University may be due to the use of both universal primers and group-specific primers, enabling the detection of bacterial presence in a larger number of samples.
The observed high prevalence of amniotic fluid bacterial colonization in our study is concerning, as previous research has linked such colonization to adverse pregnancy outcomes for both mothers and fetuses.[19]. For example, a study investigating the effects of amniotic fluid bacterial colonization on uterine activity and delivery outcomes found associations with poor cervical dilatation, response to oxytocin, and an increased risk of intrapartum infection [20]. Intrauterine infection following ascending vaginal colonization has also been implicated in causation of preterm labor, preterm births, still births, early onset neonatal sepsis among other complications[21].
Klebsiella pneumoniae, the commonest isolate in our study, has also been found to predominate amniotic fluid colonisation in PROM in studies conducted at a national referral hospital in Uganda, [6] and in Nigeria [17]. Gastrointestinal tract is a major reservoir of Klebsiella pneumoniae [22]. The close proximity of the gastrointestinal and genital tracts poses a high risk for contamination, allowing the bacteria to ascend to the cervix. This ascending colonization can lead to inflammation and subsequent rupture of the amniotic membrane[23]. Some strains of K. pneumoniae also lack the mannose content of the capsular polysaccharide that is recognized by the surface lectin of macrophages to mediate complement and antibody-independent phagocytosis. This makes them virulent and enables them to evade the body’s defence mechanisms [23].
Staphylococcus aureus, a commonly found bacterium in the human skin microbiota, emerged as the second most prevalent isolate in our study. Similar findings have been reported in other studies conducted in India [24] and a meta-analysis from China [16], indicating its predominance in amniotic fluid colonization during PROM. As a resident flora on the skin, Staphylococcus aureus can easily migrate to the genital tract [25] and subsequently ascend to the cervix. This ascent can lead to infection and inflammation of the amniotic membranes, ultimately resulting in PROM. Moreover, in cases where PROM has already occurred, the presence of Staphylococcus aureus further exacerbates the risk of complications[23]. Of particular significance, Staphylococcus aureus produces α-toxin, which facilitates the formation of biofilms. Biofilm formation serves as a protective mechanism against dehydration and immune factors such as neutrophils and macrophages. This ability of Staphylococcus aureus to form biofilms enhances its survival and proliferation within the host environment [26, 27]
In contrast to studies conducted in Canada, Australia, and America where Group B Streptococcus (GBS) was identified as the most common organism colonizing amniotic fluid in PROM [28, 29], This discrepancy may be attributed to global variations in GBS colonization among pregnant women.A systematic review encompassing 85 countries revealed significant regional variation, with higher prevalence observed in America and Canada (25%) and lower incidence in East Africa (18%) [30]. These regional disparities can be influenced by factors such as temperature variations, genetic factors, and differences in population demographics[31]. Additionally, socioeconomic factors and variations in natural immunity within different populations can play a significant role.It is worth noting that a considerable number of women in our study were referred from other healthcare facilities. As prophylaxis against GBS is part of the clinical guidelines[15], it is possible that these referred cases had already received prophylactic treatment, which could explain the lower prevalence of GBS in this study.
The bacteria isolated in our study exhibited significant resistance to the antibiotics recommended by the World Health Organization (WHO) and adopted by the Ministry of Health in Uganda for prophylaxis in PROM. All isolates were resistant to ampicillin, and the majority showed resistance to erythromycin, amoxicillin, and azithromycin. It is noteworthy that these guidelines were established based on recommendations from the ORACLE study, conducted over 20 years ago, which did not specifically focus on bacterial colonization of the female genital tract or antibiotic resistance [32]. The resistance to ampicillin observed in our study aligns with findings from other studies conducted in Nigeria, Tanzania, and Uganda [6, 17, 33]. This resistance pattern may be attributed to the overuse of these antibiotics, as penicillins are commonly prescribed for various bacterial infections [34]. The overuse of antibiotics directly contributes to the emergence of drug-resistant bacterial strains, and these resistance genes can be inherited or acquired and transferred among different species of bacteria. Additionally, antibiotics eliminate drug-sensitive competitors, providing a selective advantage for resistant bacteria to proliferate through natural selection[35]. The outdated nature of the guidelines, coupled with the alarming rates of resistance observed in this study, emphasize the importance of updating and tailoring treatment protocols to address the evolving antibiotic resistance landscape and improve patient outcomes.
The sensitivity of the isolated bacteria to certain cephalosporins was notable, with ceftriaxone demonstrating a sensitivity rate of 72.7%, and the less commonly prescribed second-generation cefuroxime exhibiting a sensitivity rate of 75%. Similar findings have been reported in a meta-analysis conducted in China, as well as studies conducted in Nigeria and Uganda [16] [17] [6]. This could be attributed to the stable β-lactam ring present in cephalosporins, which confers resistance to the action of beta-lactamases, enzymes that can inactivate certain antibiotics [36]. Overall, the highest sensitivity was observed for ciprofloxacin, at 88.6%, which aligns with findings from Nigeria where it reached 96.3% [17]. This could be attributed to ciprofloxacin having experienced a period of high resistance in the past, leading to its exclusion from many treatment protocols. As a result, its usage has been limited in recent years[37]. These findings further highlight the importance of selecting appropriate antibiotics for management of PROM based on their sensitivity profiles and considering the local resistance patterns. Continued surveillance of antimicrobial resistance could inform prescribing practices and ensure effective treatment outcomes.
Primigravidae were more likely to have cervical amniotic fluid bacteria colonisation as compared to multigravidas. Primigravidae may be at a greater risk of bacterial colonization than multigravidas due to their relatively limited interactions with the healthcare system and potential lack of exposure to medications that reduce bacterial colonization [38]. Given this increased vulnerability, primigravidae should be prioritized in the healthcare settings to avoid consequences associated with cervical amniotic fluid bacterial colonization in PROM.
Our findings revealed that obese women were more likely to exhibit cervical amniotic fluid bacterial colonization compared to women with a normal BMI. This observation aligns with previous studies that have demonstrated a link between obesity and bacterial colonization in the female genital tract[39, 40]. Notably, a significant proportion of bacteria colonizing the amniotic fluid in cases of PROM ascend from the genital tract[41]. The association between obesity and increased bacterial colonization in the genital tract may be attributed to several factors. Firstly, obesity can lead to poor genital hygiene due to excessive sweating and genital perspiration, creating an environment conducive to bacterial growth[40]. Furthermore, obese women often have higher estrogen levels resulting from peripheral aromatization, which promotes the maturation, proliferation, and accumulation of glycogen in vaginal epithelial cells. This glycogen serves as a favorable culture medium for bacterial growth[42]. As reported elsewhere [43, 44], it is crucial to prioritize the care of obese mothers by promoting good hygiene practices and implementing dietary and physical activity adjustments to mitigate the risk of cervical amniotic fluid bacterial colonization in cases of PROM.
Women who were referred in had higher odds of having cervical amniotic fluid bacteriology than those who were not referred. One plausible explanation for this observation is that a significant proportion of women with PROM in our study were referred from lower-level healthcare facilities where protocols for the accurate diagnosis and management of PROM may be lacking. Consequently, these mothers may have undergone multiple vaginal examinations and experienced a prolonged latency period before their presentation, primarily due to the challenges associated with referral transportation in our setting [45, 46]. However, the effect of the number of vaginal examinations was not studied in this study. This may have resulted into this residual confounding from this unstudied factor. Future studies should examine the specific factors related to referral and their impact on colonization risk in order to improve diagnosis and management strategies for this subgroup of women.
Limitations
Our study had some limitations that should be considered. Firstly, we were unable to conduct gene sequencing on the PCR-positive samples, which restricted our ability to identify and characterize specific bacterial isolates present in the samples. This information could have provided valuable insights into the microbial composition and potential virulence factors associated with cervical amniotic fluid bacterial colonization. Secondly, our study was conducted at a single site, which may limit the generalizability of our findings to other healthcare settings. The characteristics and practices at our study site may not fully represent the diverse populations and variations in bacterial colonization rates observed in different regions. Additionally, the lack of participant follow-up in our study prevented us from assessing important outcomes and implications related to bacterial colonization, such as maternal sepsis and neonatal infections. Future studies with longer follow-up periods are needed to evaluate the clinical outcomes and risks associated with cervical amniotic fluid bacterial colonization.
Despite these limitations, our study benefited from the use of highly sensitive PCR diagnostic testing, which improved the accuracy of detecting bacterial colonization in addition to traditional culture methods. This enhanced sensitivity strengthens the validity of our findings regarding the prevalence of bacterial colonization in women with PROM.